From the Department of Biochemistry, School of Pharmacy, University of Barcelona, Avenida Diagonal 643, 08028 Barcelona, Spain and the § Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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The metabolic pathways specified by the
glc and ace operons in Escherichia
coli yield glyoxylate as a common intermediate, which is acted on
by two malate synthase isoenzymes: one encoded by glcB and
the other by aceB. Null mutations in either gene exhibit no
phenotype, because of cross-induction of the ace operon by glycolate and the glc operon by acetate. In this study, the
regulation of the glc operon, comprising the structural
genes glcDEFGB, was analyzed at the molecular level. This
operon, activated by growth on glycolate, is transcribed as a single
message and is under the positive control of GlcC encoded by a
divergent gene. Expression of the glc operon is strongly
dependent on the integration host factor (IHF) and is repressed by the
global respiratory regulator ArcA-P. In vitro gel-shift
experiments demonstrated direct binding of the promoter DNA to IHF and
ArcA-P. Mutant analysis indicated that cross-induction of the
glc operon by acetate is mediated by the GlcC protein that
recognizes the compound as an alternative effector. The similar pattern
of regulation of the Glc and Ace systems by IHF and ArcA-P ensures
their effective cross-induction.
Glyoxylate is an important intermediate of the central microbial
metabolism in the "glyoxylate bypass," required when acetate or
fatty acids are the main carbon and energy source (1). Glyoxylate is
also generated from glycolate or purine degradation in
Escherichia coli (2, 3) and is subsequently converted into
malate (Fig. 1). Nevertheless, a
constitutive glyoxylate reductase activity has been reported to convert
glyoxylate back to glycolate (2).
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Metabolic map of the pathways converging on
glyoxylate. Glycolate, acetate, and purine degradation pathways
are shown only with the enzymes relevant to this work.
There are two isoenzymes of malate synthase in Escherichia coli, malate synthase A (MSA)1 for growth on acetate and malate synthase G (MSG) for growth on compounds metabolized via glycolate/glyoxylate (4, 5). These two enzymes are distinguishable by thermal stability and kinetic properties (5) and recently have been shown to have significantly different amino acid sequences (6). The genes of the glycolate pathway that encode glycolate oxidase (GOX) (glcDEF) and MSG (glcB) are located in a cluster at 64.5 min. Their expression is induced by growth on glycolate and controlled by the gene glcC (6, 7). Disruption of this gene resulted in a phenotype that indicated the product to be an activator protein (GenBankTM accession number L43490; Ref. 7). The genes of the acetate pathway that encode isocitrate lyase (ICL) (aceA), MSA (aceB), and isocitrate dehydrogenase kinase/phosphatase (aceK) are clustered and transcriptionally repressed by the products of iclR and fadR (8, 9). The formation of the complex between the IclR repressor and the operator/promoter region has been reported to be impeded by phosphoenolpyruvate (8).
Results from different experiments point to the cross-induction of
glc and ace operons. On the one hand, growth on
glycolate induced an ICL structurally and functionally
indistinguishable from that induced by acetate (10). On the other hand,
it appears that aceB mutants failed to grow on acetate only
if MSG was not available as a back up enzyme in glc mutants
(4). So far, however, the recruitment mechanism of MSG for the acetate
pathway and the putative involvement of MSA in glycolate metabolism
have not been documented. Here we provide direct experimental evidence
for the cross-induction of these two systems as well as the molecular basis for the acetate-induced glc expression.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Phages-- All the strains used were E. coli K-12 derivatives. The genotype and sources of the bacterial strains are given in Table I. Strain JA159 was obtained by inserting a chloramphenicol acetyltransferase (cat) cassette in the ClaI restriction site of gene glcB as described previously (7). Other strains were constructed by P1 transduction (11). Transductants that lost the glu-1 or glc mutations were selected on glucose or glycolate, respectively, and transductants that incorporated the arcA mutation by their sensitivity to O-toluidine blue (12).
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Cell Growth, Preparation of Cell Extracts, and Enzyme
Activities--
Cells were grown and harvested as described previously
(13). For aerobic growth, carbon sources were added to a basal
inorganic medium (13) at 60 mM carbon concentration and for
anaerobic growth at 120 mM. Casein acid hydrolysate (Caa)
was used at 0.5% for aerobic or at 1% for anaerobic cultures and for
growth of transformed cells. Oleic acid was provided at 5 mM in the presence of Brij 58 (5 mg/ml). For anaerobic
respiration nitrate was added to the cultures at 20 mM
concentration. When required, the following antibiotics were used at
the indicated concentrations: ampicillin, 100 µg/ml; tetracycline,
12.5 µg/ml; chloramphenicol, 30 µg/ml; and kanamycin, 50 µg/ml.
5-Bromo-4-chloro-3-indolyl--D-galactoside and
isopropyl-
-D-thiogalactoside were used at 30 and 10 µg/ml, respectively. For
-galactosidase assays, the cells were
allowed to double 5-6 times to an OD600 of 0.5 for aerobic
cultures or 0.25 for anaerobic cultures.
Cell extracts were prepared as described previously (14). GOX and MSG
activities were determined as described by Pellicer et al.
(7), and -galactosidase activity was assayed by hydrolysis of
O-nitrophenyl-
-D-galactopyranoside and
expressed as Miller units (11). Values reported in this work are a
representative set of at least three separate experiments performed in
duplicate. Protein concentration was determined by the method of Lowry
(15) using bovine serum albumin as standard.
Immunological Techniques-- Antisera against MSG were raised in New Zealand White rabbits as described previously (16) using the purified enzyme as antigen (6). Quantitative immunoelectrophoresis was performed as described by Laurell (17). The agarose gel contained 1.3% specific globulins; 65 µg protein in 10 µl cell extract was applied to each of the wells. The specificity of this preparation was confirmed by the lack of immunoprecipitate in cell extracts of strains JA154 and JA159.
DNA Manipulation and Sequencing-- Plasmid DNA was routinely prepared by the boiling method (18). For large scale preparation, a crude DNA sample was subjected to purification on a column (Qiagen GmbH, Düsseldorf, Germany). Other DNA manipulations were performed essentially as described by Sambrook et al. (19). The DNA sequence was determined by using the dideoxy-chain termination procedure of Sanger et al. (20), with double-stranded plasmid as the template. Sequencing gel compressions were resolved as described elsewhere (7).
Isolation of RNA, Northern Blot Hybridization, and Primer
Extension--
For preparation of total RNA, cells of a 25-ml culture
grown to an A650 of 0.5 were collected by
centrifugation at 5,000 × g and processed according to
Belasco et al. (21). For primer extension analysis the RNA
was prepared with a Qiagen RNeasy Total RNA kit. Northern blot
hybridization was performed with each RNA sample (10 µg) by the
procedure described by Moralejo et al. (22). For the
determination of the 5'-end of the structural and the regulatory genes,
the following oligonucleotides were used as primers:
5'-GGTCGACATCGGGTAAAGC-3' (complementary to an internal region within
glcD) and 5'-TGACCGACCTTCAGTACCCG-3' (complementary to a
glcC internal sequence). The reaction was performed with 50 µg of total RNA at 37 °C for 30 min with 200 units of M-MLV reverse transcriptase (Life Technologies, Inc.) and
[-35S]thio-dATP (>1,000 Ci/mmol; Amersham Pharmacia
Biotech), and this was followed by a 30-min chase with all four
nucleotides (at 1 mM each) (23). As a reference,
double-strand sequence reactions were performed with the same primers.
Construction of lacZ Fusions and Deletions of the glcD Promoter
Sequences--
To create operon fusions, DNA fragments of the
5'-upstream region of each glc gene (Fig.
2) were cloned into plasmid pRS550 or
pRS551 (24). These plasmids carried a cryptic lac operon and
genes that confer resistance to both kanamycin and ampicillin. Recombinant plasmids were selected, after transformation of strain XL1Blue, as blue colonies on LB plates containing
5-bromo-4-chloro-3-indolyl--D-galactoside, ampicillin,
and kanamycin, and plasmid DNA was sequenced by using the M13 primer to
ensure that the desired fragment was inserted in the correct
orientation. Merodiploids were obtained by transferring the fusions as
single copies into the trp operon of the E. coli strain TE2680 as described by Elliot (25). The transformants were
selected for kanamycin resistance and screened for sensitivity to
ampicillin and chloramphenicol. P1 vir lysates were made to transduce the fusions into strain MC4100.
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Fragments containing different 5'-deleted sequences of the
glcD promoter region were created by polymerase chain
reaction (PCR). The 18-mer oligonucleotide 5'-TAAAGCGCCATCAAGACG-3',
identical to the sequence between +77 and +94 of the non-coding strand
of gene glcD with additional bases at the 5'-end to give an
EcoRI site at one end of the PCR product, was used as the
constant primer. The partner primers extended from the following
positions: 247 to
230,
192 to
177,
184 to
159,
177 to
159,
170 to
153,
155 to
139,
103 to
86,
88 to
71,
54 to
37, and
27 to
10, all bearing additional nucleotides at
the 5'-end to generate a BamHI site at the other end of the
PCR product. After digestion with BamHI and
EcoRI, the PCR products were cloned into the pRS550, and the
corresponding recombinant plasmids were used to construct single copy
fusions in strain MC4100 as described above. Among the fragments
described above, the three obtained with the partner primers,
247 to
230,
192 to
177, and
170 to
153, were also cloned into pRS551
yielding sequences of the glcC promoter deleted at the
3'-end and fused to lacZ. Single copy fusions of these constructs were also obtained in strain MC4100.
Purification and Phosphorylation of His6-ArcA
Protein--
Purification of the His-tagged ArcA protein from
isopropyl-1-thio--D-galactopyranoside-induced E. coli M15 cells transformed with pREP4 and pQE30ArcA, by nickel
chelate affinity chromatography using the nickel-nitrilotriacetic acid
resin (Qiagen), was performed as described previously (26). Purified
His6-ArcA (50 µg/ml) was phosphorylated with 50 mM disodium carbamyl phosphate (Sigma) at 30 °C for
1 h.
DNA Binding Studies--
For the binding studies with ArcA-P or
IHF, the 360-bp DNA fragment containing the glcD promoter
sequence (247 to +94) was obtained by PCR with primers
GLCBam (5'-CGCGGATCCGTTCGATACTCTCTGCAACC-3') and
GLCEco (5'-GGGGAATTCTAAAGCGCCATCAAGACG-3') using
plasmid pTP25 (7) as a template. For binding studies with GlcC, the
113-bp DNA fragment containing the glcD promoter sequence
(
247 to
134) was obtained by PCR with primers GLCBam and
GLC P (5'-GGGGAATTCGTGCGTGTTTTGCGAG-3'). PCR-amplified
products were purified from acrylamide gels and labeled by using T4
polynucleotide kinase and [
-32P]ATP (3,000 Ci/mmol,
NEN Life Science Products). End-labeled fragments for DNase I
footprinting assays were generated by digestion of the labeled DNA
fragments with a restriction endonuclease that cleaves (uniquely) close
to either of the termini. The short 32P-labeled,
double-stranded DNA fragments generated by restriction enzyme digestion
and non-incorporated nucleotides were removed by sequential ethanol precipitation.
Electrophoretic mobility shift assays for GlcC or IHF were performed using crude extracts obtained as described by Nunoshiba et al. (27). Acrylamide gels containing 10% glycerol and 0.5× TBE buffer were run at 4 °C (28). Protein samples were mixed with 32P-end-labeled DNA substrates (~2.5 nM final concentration, ~10,000 to 25,000 cpm) in a 20-µl reaction volume containing 10 mM Tris·HCl (pH 7.5), 75 mM KCl, 10% glycerol, and 2 mM dithiothreitol. Poly(dI-dC) was used as nonspecific competitor. After incubation for 15 min at 25 °C, 1/6th volume of a 6× gel loading buffer (28) was added, and the mixture was loaded directly onto pre-run gels.
Electrophoretic mobility shift assays with purified ArcA-P were performed in acrylamide gels run at 4 °C using 1× TBE buffer (28). Protein samples were combined with 32P-end-labeled DNA substrates (~2.5 nM final concentration, ~10,000 to 25,000 cpm) in a 30-µl reaction volume containing 100 mM Tris·HCl (pH 7.4), 100 mM KCl, 10 mM MgCl2, 10% glycerol, and 2 mM dithiothreitol. After incubation for 30 min at 25 °C, 1/6th volume of a 6× gel loading buffer (28) was added, and the mixtures were loaded directly onto pre-run gels. Where indicated, sheared (100-600 bp) herring sperm DNA (Promega Corp.) was included as a nonspecific competitor at a concentration equivalent to a 500-fold molar excess over the labeled DNA-binding substrate.
Analysis of DNA binding by nuclease protection assays was performed as
described previously (26). The 360-bp DNA fragment, 5'-end-labeled with
32P was digested with BamHI or EcoRI.
Protein samples were combined with 32P-end-labeled DNA
substrates (~2 nM final concentration, ~10,000 cpm) in
50-µl reaction mixtures containing 50 mM Tris·HCl (pH 8.0), 100 mM KCl, 12.5 mM MgCl2, 1 mM Na2EDTA, 20% glycerol, 1 mM
dithiothreitol. The reaction mixtures were incubated for >10 min on
ice, 50 µl of 5 mM CaCl2, 10 mM
MgCl2 was added, and the incubation was continued for 1 min
at room temperature. Then 3 µl (0.15 units) of RQ1 RNase-free DNase I
(Promega) was added, and following a further minute at room
temperature, the nucleolytic reaction was terminated by addition of 90 µl of 200 mM NaCl, 30 mM Na2EDTA,
1% SDS, 100 µg of yeast RNA per ml. After the phenol/chloroform extraction, DNA products were recovered by ethanol precipitation, and
resuspended in 0.1 M NaOH/formamide (1:2 v/v), 0.1% xylene cyanol, 0.1% bromphenol blue. Reaction mixtures were heated at 95 °C for 2 min prior to loading on 5% polyacrylamide wedge-shaped sequencing gels.
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RESULTS |
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Experimental Evidence of the Cross-induction of ace and glc Operons-- Retention of growth abilities on acetate and glycolate by mutants defective in either aceB (strain JA166) or glcB (strain JA159) first suggested the cross-induction of these two systems. Consistent with this notion, a double aceB glcB mutant (strain JA168) failed to grow on both acetate and glycolate. On the other hand, mutations abolishing ICL activity (strains JA165 and JA167) only prevented growth on acetate, and mutations abolishing GOX activity (strain JA155) prevented only growth on glycolate, despite the presence of both MSA and MSG.
Further evidence for the cross-induction was obtained by assaying MSA, MSG, and GOX activities on crude extracts of the wild-type strain MC4100 grown on acetate or glycolate. Basal levels of these activities on casein acid hydrolysate were found to be in the range of 20 milliunits/mg for MSA and MSG and <5 milliunits/mg for GOX. Glycolate induced MSA activity at a level of 80 milliunits/mg, one-half of the 170 milliunits/mg obtained when the cell grew on acetate. MSG and GOX were coordinately induced to levels of 450 and 40 milliunits/mg, respectively, on cultures grown on glycolate, whereas growth on acetate induced MSG levels to significantly lower values (70 milliunits/mg). The 6-fold lower level of glc induction in this carbon source, as indicated by MSG activities, brings GOX activity below limits of detection (<5 milliunits/mg). Nevertheless, acetate-induced expression of genes encoding GOX subunits was evidenced by Northern experiments using RNA preparations obtained from strain MC4100 grown on acetate. Levels of MSG induction in the different conditions were confirmed by immunochemical detection of the MSG protein (not shown).
Transcriptional Organization of the glc Gene Cluster--
To
understand better the mechanisms of the ace and
glc cross-induction, we proceeded to determine the
transcriptional units of the glc gene cluster. Previous
Northern blot experiments with the wild-type strain MC4100 failed to
detect a polycistronic mRNA (7), possibly because of a message
decay. To circumvent such a possibility, we grew an RNase E
temperature-sensitive mutant strain CH1828 and its isogenic parent
CH1827 on Caa plus glycolate to compare the results of Northern blots.
Only RNA preparations of mutant strain CH1828 grown at restrictive
temperature showed an mRNA of 7.5 kb corresponding to the
full-length transcript of the glc system (Fig.
2A). The same polycistronic mRNA was detected using
either a glcD or glcB probe. Transcription of the
genes glcDEFGB as a single unit is supported by the
properties of five lacZ fusions corresponding to each of the
glc genes (Fig. 2B). These operon fusions were
transferred to strain MC4100. Of the five resulting merodiploids grown
on Caa either in the presence or in the absence of glycolate, only the
one bearing the glcD-lacZ fusion expressed a
glycolate-inducible -galactosidase (Fig.
3A). By contrast, the other
four fusions exhibited neither significant basal nor inducible activity
(not shown). Furthermore,
(glcEFGB), containing a
3'-fragment of glcD (Fig. 2B), also exhibited no activation of transcription by glycolate. The same pattern of expression was obtained in acetate-grown cells. These observations indicated that the only functional promoter for the glc
structural genes is located upstream of glcD.
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The expression of (glcC-lacZ) corresponding to the
regulator gene, in contrast to that of
(glcD-lacZ), was
repressed by the presence of glycolate (Fig. 3A).
Mapping of the mRNA 5'-End for the Structural and Regulator
Transcriptional Units--
The 5'-end of the structural genes was
determined by primer extension analysis. Total mRNA from strain
MC4100 grown in the presence of glycolate was obtained. For the primer
extension reaction a primer complementary to a region within
glcD (positions +52 to +33 of the coding region) was used,
and a single putative 5'-end was determined (Fig.
4B). The 5'-end was thus
located 55 bp upstream of the ATG codon. The putative 5'-end, position
+1 in Fig. 4A, is preceded by a promoter sequence (35,
TAGACG;
10, TAATAA, with a spacing of 17 bp) that conforms relatively
well to the
70 consensus sequence.
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The 5'-end of glcC was also determined by the same method
using a primer complementary to an internal region of glcC
(positions +86 to +65 of the coding region). An extended product was
detected giving a 5'-end located 66 bp upstream of the glcC
start codon (Fig. 4B). Relatively conserved 10 and
35
boxes were found (Fig. 4A), as expected for the weak
promoter activity of regulatory genes.
The glc Operon Is Under the Control of at Least Three
Transcriptional Regulators--
The role of the glcC gene
product as activator of the glc operon was assessed by
assaying -galactosidase in strain JA154 glcC::cat
(glcD-lacZ). The insertion
of cat completely abolished the expression of the fusion,
regardless of whether the cells were grown on Caa in the presence or
absence of glycolate (Fig. 3A). Expression analysis of the
regulator gene glcC was performed in parallel cultures of
strain JA154 bearing the
(glcC-lacZ) (Fig.
3A). Note that in this glcC mutant, expression of
-galactosidase from this promoter is not reduced by the presence of glycolate.
Involvement of IHF in the expression of the glc operon was
tested by assaying -galactosidase in strain JA162
himA::cat
(glcD-lacZ) lacking one of
the IHF subunits. The insertion of cat completely abolished
the expression of the fusion, regardless of whether the cells were
grown on Caa in the presence or absence of glycolate (Fig.
3A). Similar results were observed in strain JA163
himD3::cat
(glcD-lacZ) lacking the
other IHF subunit (data not shown). As expected, IHF mutants were
unable to grow on glycolate as the sole source of carbon and energy.
Parallel experiments with strain JA162 himA::cat
(glcC-lacZ) indicated that the expression of the
regulatory gene was unaffected by the cat insertion (Fig. 3A). Gel retardation experiments with a promoter fragment
encompassing positions
247 to +94 (see "Experimental Procedures")
displayed a major retarded complex with crude extracts of
wild-type strain. No such complex appeared when an extract from a
himA mutant was used, indicating that IHF was the protein
responsible for the retardation of the glcD promoter DNA
(Fig. 5A).
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To test whether glc operon was regulated by the availability
of oxygen and other electron acceptors, the expression of
(glcD-lacZ) was assayed in cells grown on Caa in the
presence of glycolate with or without the addition of the efficient
electron acceptor nitrate. The expression of
(glcD-lacZ)
was highest during aerobic cell growth, whereas it was almost
completely abolished in the absence of oxygen (Fig. 3B).
Anaerobically, nitrate raised the activity to the aerobic levels (Fig.
3B), whereas it exerted no effect on aerobic expression (not
shown). These results, together with the fact that the addition of 15 mM pyruvate to anaerobic cultures promoted a 3-fold
repression of
(glcD-lacZ) expression (29), suggested that
the glc operon was controlled by the two-component system
ArcB/ArcA. This control is expected to be exerted only on the promoter
of the operon comprising the structural genes, since the absence of
oxygen does not change the
(glcC-lacZ) expression (data
not shown). To verify this expectation, an arcA null
mutation was introduced in strain MC4100, yielding strain JA164. The
expression of
(glcD-lacZ) in this genetic background was
no longer repressed under fermentative conditions, indicating that ArcA
acts as an anaerobic repressor of the glc operon (Fig.
3B).
To analyze the interaction of ArcA and ArcA-P with the glc promoter region, gel retardation assays were carried out using purified His6-ArcA and His6-ArcA-P generated by treatment of the former with carbamyl phosphate. The results indicated that the phosphorylation of ArcA significantly enhanced its DNA binding activity (Fig. 6A). Increasing the concentration of ArcA-P in the incubation mixture resulted in the appearance of further retarded species, indicating multiple binding and/or oligomerization of ArcA-P within the glc promoter region.
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The binding of His6-ArcA and His6-ArcA-P to the
glc promoter region was also analyzed by DNase I
footprinting. No discrete regions of protection could be observed with
His6-ArcA, whereas clear DNase I footprints were seen with
His6-ArcA-P. It is noteworthy that within the protected
regions there are highly protected segments as follows: from positions
45 to +31 (ArcA site I) and from
106 to
161 (ArcA site II)
relative to the 5'-end of glcD gene (Fig. 6B).
Protection of the two ArcA sites was observed on both strands. These
large protected regions again suggested ArcA-P binding to multiple
sites and/or oligomerization of the regulator protein molecule.
Deletion Analysis of the glcD Promoter-- To locate the cis-acting elements required for regulation of the glcD promoter, different 5'-deletions were fused to the lacZ reporter gene and introduced as single copy fusions in MC4100 background.
Ten constructions labeled by the 5' terminus were analyzed by
-galactosidase expression in cultures grown aerobically on Caa,
either in the presence or in the absence of glycolate. Results presented in Fig. 7 show that full
induction of
(glcD-lacZ) by glycolate required sequences
up to position
184. The construct f-177 showed a 40% reduction in
activity levels, whereas construct f-170 totally lost the ability for
expression. These results therefore point to an upstream activator site
(UAS) for GlcC binding (
184 to
170). Moreover, the location of this
site between the 5'-end and the ATG start codon of glcC
could also explain the reduced expression of the regulator gene
glcC during growth in the presence of glycolate. This was
further supported by the results obtained with three transcriptional
fusions in the opposite orientation which expressed the glcC
promoter activity (r-247, r-192, and r-170 in
Fig. 7). Absence of the UAS element in these constructs caused enhanced
activation of glcC transcription. Hence, while activating
the transcription of the structural genes, GlcC exerts an autogenous
repression on its own gene. Interaction of GlcC with the UAS element
was assessed by gel retardation experiments performed with a 113-bp
fragment of glcD promoter (positions
247 to
134)
containing this element. Whereas retardation was observed with an
extract of the wild-type strain, no effect was seen with that of the
glcC mutant (Fig. 5B).
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Since it is likely that the IHF effect is mediated by sequences
downstream of position 170, a computer search for IHF sites (30) was
undertaken. Probable consensus sequences were found between positions
128 to
93 in the glcD coding strand and
142 to
95 in
the complementary strand (Fig. 4A). The construct f-54, lacking a A/T-rich segment likely to facilitate bending (positions
61
to
69) but retaining the
35 and
10 promoter sequences, allowed a
(glcD-lacZ) expression at 10% of the normal
level, and this expression was independent of GlcC. The same result was obtained when this construct was expressed in a himA mutant
(not shown) indicating that the IHF effects are mediated by sequences upstream of position
54. This basal expression was lost in the construct f-27 missing the
35 sequence for RNA polymerase binding.
Induction of the glc Operon by Acetate Is Mediated by the Action of
GlcC Protein on the glcD Promoter--
The introduction of a
cat insertion in glcC absolutely abolished
induction of glc by acetate, indicating that the
cross-induction was mediated by the glcC gene product (not
shown). Moreover, when strain MC4100 bearing the
(glcD-lacZ) was transformed with a pBS derivative plasmid
containing the 400-bp SalI fragment flanking the
glcD promoter, expression from the chromosomal fusion was reduced by about 80%. This diminution, produced either in the presence
of glycolate or acetate as inducers, can be explained by GlcC titration
by the high copy number of glcD promoter. Consistent with
this interpretation, the induced activity levels were high (70% of the
control level) when sequences upstream of position
170 were
eliminated from the construct.
To analyze whether acetate induces the glc operon expression
by itself or via glyoxylate formation, the
(glcD-lacZ) was introduced in mutant strains
JA165 (ICL-deficient), JA166 (MSA-deficient), and JA168 (lacking both
MSA and MSG).
-Galactosidase assay and immunological quantification
of MSG were performed on cultures of these strains growing on Caa
0.05% in the presence of acetate or glycolate, with strain MC4100
(glcD-lacZ) serving as control (Fig.
8). Acetate induction in an ICL-deficient
mutant suggested that the effector molecule responsible for the
cross-induction could be acetate itself or any derived metabolite
leading to isocitrate formation (Fig. 8). To check if acetate itself
acted in the cross-induction, strain JA165
(glcD-lacZ)
was grown on 0.05% Caa in the presence of oleate whose metabolism
provide acetyl-CoA but not acetate. Levels of induction in the presence
of oleate (180 Miller units) were similar to those obtained in the
absence of this fatty acid and were about less than 50% those obtained
in the presence of acetate (450 Miller units). These results indicate
that acetate but not acetyl-CoA is the effector molecule.
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In strain JA166 (glcD-lacZ), lacking MSA, the
pool of unmetabolized glyoxylate is expected to increase during growth
on acetate. In this strain, glyoxylate itself or glycolate formed by
glyoxylate reduction enhanced
(glcD-lacZ)
expression and the level of MSG synthesis by 2.5-fold. In strain JA168
(glcD-lacZ), lacking both MSG and MSA, the
intracellular glyoxylate pool was even higher, and the expression of
lacZ by acetate increased 5-fold. The increased promoter
activity in Caa-grown cells suggests that glyoxylate may be formed from
amino acid degradation. Results of immunoassay of MSG levels in all of
the mutants grown under various conditions (Fig. 8) matched the results
of
(glcD-lacZ) expression.
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DISCUSSION |
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It is tempting to speculate that the presence of redundant malate synthases and the cross-induction of the glc and ace operons evolved as a safeguard against toxic accumulation of the chemically reactive glyoxylate. The hypothesis is consistent with the enhanced cross-induction resulting from glyoxylate accumulation in mutants lacking either of the malate synthase isoenzymes.
Molecular characterization of the cross-induction required fine structure analyses of these operons. The ace operon has been well studied (31, 32), and its inducer has been identified to be phosphoenolpyruvate (8). Thus, the generation of this inducer from glycolate via the D-glycerate pathway (2) would explain cross-induction of the ace operon by glycolate. Here we report a fine structure study of glc operon and its regulation by glycolate and acetate. Promoter deletion analysis has permitted us to define a cis-acting element (labeled UAS) that has been shown to interact with glcC gene product in the presence of glycolate. This regulator protein functions simultaneously as glcDEFGB activator and glcC repressor. When the glc system is cross-induced it appears that acetate or a derivative serve as an alternative effector. The use of an ICL (aceA) mutant showed that glyoxylate formation from acetate was not required for induction. The inability of oleate, which yields acetyl-CoA but not acetate, to induce the glc system in this ICL mutant corroborates the role of acetate as inducer molecule.
In the context of the cross-induction, several features of the
glc promoter are of interest. Anaerobic repression of
glc by ArcA-P is consistent with the fact that both acetate
and glycolate metabolisms take place aerobically (33). Acetate cannot
be utilized as a carbon source without the aerobic Krebs cycle, and the
ace operon has been suggested to be a target for the Arc
modulon (34). On the other hand, glycolate oxidase is absolutely
dependent on the aerobic electron transport chains of the bacterial
membrane. ArcA mutants, footprinting, and gel-shift experiments indeed
demonstrated a functional control of glc by the Arc system.
In this regard it might be pointed out that the ArcA-P-protected
regions contained three sequences with high similarity (7:10 agreement)
to the consensus proposed by Lynch and Lin (26): site I from positions
7 to
16 and site II from positions
102 to
111 and from
positions
110 to
119 (Fig. 4A). Although the proposed
consensus has recently been questioned, no amendment was offered (35).
On the other hand, the two consensus sequences (10:10 agreement) in
aldA promoter have been useful in predicting the in
vitro site of ArcA-P binding and in vivo regulation as
demonstrated by site-directed
mutagenesis.2 The overlap of
site I with the
10 promoter sequence would explain the
transcriptional repression of glc operon in anaerobic
conditions. Hypersensitivity to DNase I at several positions within the
ArcA-P-protected regions suggests that ArcA-P promoted DNA bending.
The proposed hairpin structure for this promoter is supported by the
absolute dependence of its activation on IHF. It has to be underscored
that in this system the IHF effect is not mediated by a repression in
the expression of the regulator gene glcC (see Fig.
3A). Binding of IHF to sequences between the distant
specific activator site (UAS) and the 35 and
10 consensus sequences
for RNA polymerase binding has been reported for several
bending-dependent promoters (36, 37). In most of the
reported cases the modulating effect of IHF on gene expression is
modest (changes of 2-5-fold). Strong roles of IHF have been reported
for only a few systems, including nifHDK,
narGHJI, or tdc (38). By contrast, glc
expression is absolutely IHF-dependent. IHF has also been
reported to activate the ace operon (39). Thus, the
metabolic role of cross-induction is made possible by the fact that
both ace and glc operons are similarly controlled
by Arc and IHF.
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ACKNOWLEDGEMENTS |
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We thank Simon Lynch for critical reading of the manuscript and Robin Rycroft for editorial assistance.
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FOOTNOTES |
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* This research was supported in part by Grant PB97-0920 from the Dirección General de Investigación Científica y Técnica, Madrid, Spain, by the help of the "Comissionat per Universitats i Recerca de la Generalitat de Catalunya," and by U. S. Public Health Service Grants GM-39693 and GM-40993 from the National Institutes of Health.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.
Recipient of a predoctoral fellowship from the Generalitat de Catalunya.
¶ To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, School of Pharmacy, University of Barcelona, Diagonal, 643, 08028 Barcelona, Spain. Tel.: 34-93-402 45 21; Fax: 34-93-402 18 96; E-mail: baldoma{at}farmacia.far.ub.es.
The abbreviations used are: MSA, malate synthase A; IHF, integration host factor; MSG, malate synthase G; GOX, glycolate oxidase; ICL, isocitrate lyase; cat, chloramphenicol acetyltransferase; Caa, casein acid hydrolysate; PCR, polymerase chain reaction; UAS, upstream activator site; bp, base pair.
2 M. T. Pellicer, A. S. Lynch, P. De Wolf, D. Boyd, J. Aguilar, and E. C. C. Lin, manuscript in preparation.
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