Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli - PubMed (original) (raw)
Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli
Laura Aguilera et al. J Bacteriol. 2008 Apr.
Abstract
The lldPRD operon of Escherichia coli, involved in L-lactate metabolism, is induced by growth in this compound. We experimentally identified that this system is transcribed from a single promoter with an initiation site located 110 nucleotides upstream of the ATG start codon. On the basis of computational data, it had been proposed that LldR and its homologue PdhR act as regulators of the lldPRD operon. Nevertheless, no experimental data on the function of these regulators have been reported so far. Here we show that induction of an lldP-lacZ fusion by L-lactate is lost in an Delta lldR mutant, indicating the role of LldR in this induction. Expression analysis of this construct in a pdhR mutant ruled out the participation of PdhR in the control of lldPRD. Gel shift experiments showed that LldR binds to two operator sites, O1 (positions -105 to -89) and O2 (positions +22 to +38), with O1 being filled at a lower concentration of LldR. L-Lactate induced a conformational change in LldR that did not modify its DNA binding activity. Mutations in O1 and O2 enhanced the basal transcriptional level. However, only mutations in O1 abolished induction by L-lactate. Mutants with a change in helical phasing between O1 and O2 behaved like O2 mutants. These results were consistent with the hypothesis that LldR has a dual role, acting as a repressor or an activator of lldPRD. We propose that in the absence of L-lactate, LldR binds to both O1 and O2, probably leading to DNA looping and the repression of transcription. Binding of L-lactate to LldR promotes a conformational change that may disrupt the DNA loop, allowing the formation of the transcription open complex.
Figures
FIG. 1.
Organization of transcriptional regulatory elements of the lldPRD operon. (A) The lldPRD promoter sequence is numbered relative to the 5′ end determined in this study, which is shown by an arrowhead labeled “+1.” The −10 and −35 promoter sequences are underlined, and the ribosome binding site (RBS) and predicted ATG start codon (in bold) are indicated. The two previously proposed promoters, i.e., the basal P1 and inducible P2 (13) promoters, and the corresponding transcriptional initiation sites (open triangles) are also indicated above the nucleotide sequence. The predicted PdhR and LldR binding sites (13, 23) are named the O1 and O2 operator sites, respectively. (B) Identification of lldPRD 5′ end by sequencing across ligation sites of 5′-RACE products. Chromatograms display the sequences at ligation sites of typical cloned 5′-RACE products derived from transcripts obtained from MC4100 cells grown in CAA or in
l
-lactate. Arrows indicate the transcription initiation site.
FIG. 2.
Analysis of transcriptional fusions to identify functional promoters in the lldPRD genetic system. The extension and direction of the lld genes, lldP (encoding
l
-lactate permease), lldR (encoding the regulatory protein), and lldD (encoding
l
-lactate dehydrogenase), are indicated by open arrows. The transcriptional start site (position +1) and the two putative operator sites, O1 and O2, are indicated upstream from these genes. Fragments fused to lacZ for testing of promoter function are shown below and are numbered relative to the transcriptional start site. These fragments were fused to lacZ and introduced as single-copy fusions in the genetic background of strain MC4100. The values for β-galactosidase activity under the different growth conditions are indicated in the table on the right and expressed as means ± SD.
FIG. 3.
Effects of deletions, mutations, or changes in O1-to-O2 helical phasing on lldPRD expression. The two LldR binding sites (O1 and O2 operators) are represented by black boxes in the diagram shown at the top. The gray box corresponds to the −35 promoter sequence identified in this study. The different constructs are shown below the top diagram and numbered at the left side. Mutations in either O1 or O2 are indicated by hatched boxes, and the mutation of the −35 promoter sequence is marked by an asterisk. The 5-bp or 10-bp insertions between both operator sites are indicated at the bottom (lines 7 and 8). These fragments were fused to lacZ and introduced as single-copy fusions in the genetic background of strain MC4100. Values for β-galactosidase activity under inducing or noninducing conditions of growth are indicated in the table on the right and expressed as means ± SD.
FIG. 4.
β-Galactosidase activities of the Φ(lldP-lacZ) transcriptional fusion in different genetic backgrounds. Cells were grown aerobically in CAA (black bars) or in CAA plus 20 mM
l
-lactate (white bars). Activity values are expressed as means ± SD.
FIG. 5.
Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated DIG-labeled DNA probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).
FIG. 6.
Characterization of LldR binding to O1 and O2 operators. (A) Electrophoretic mobilities of the LldR-O1 and LldR-O2 complexes formed at increasing concentrations of protein. DIG-labeled DNA probes (for the region of each probe, see Fig. 4) were incubated at 30°C for 15 min with the indicated amounts of LldR and subjected to PAGE. (B) Effects of mutations in O1 and O2 operator sites on LldR binding. Mutations introduced by site-directed mutagenesis into the O1 or O2 palindromic sequence are shown in bold below the corresponding wild-type sequence. The arrows indicate the inverted repeat present in each operator site. The corresponding DIG-labeled fragments were added to binding mixtures containing the indicated amounts of LldR and incubated and processed as described above.
FIG. 7.
Effect of
l
-lactate on cleavage of the fusion protein MBP-LldR by factor Xa. Proteins were separated by sodium dodecyl sulfate-10% PAGE and stained with Coomassie blue. Lane 1, MBP-LldR fusion protein eluted from the amylose column with 10 mM maltose before cleavage with factor Xa; lane 2, MBP-LldR digestion products after an overnight incubation with factor Xa in the presence of 10 mM
l
-lactate; lane 3, MBP-LldR digestion products after an overnight incubation with factor Xa in the absence of 10 mM
l
-lactate. Molecular masses of the markers are indicated on the left.
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