Identification of the Set of Genes, Including Nonannotated morA , under the Direct Control of ModE in Escherichia coli

Abstract

dModE is the molybdate-sensing transcription regulator that controls the expression of genes related to molybdate homeostasis in Escherichia coli. ModE is activated by binding molybdate and acts as both an activator and a repressor. By genomic systematic evolution of ligands by exponential enrichment (SELEX) screening and promoter reporter assays, we have identified a total of nine operons, including the hitherto identified modA, moaA, dmsA, and napF operons, of which six were activated by ModE and three were repressed. In addition, two promoters were newly identified and direct transcription of novel genes, referred to as morA and morB, located on antisense strands of yghW and torY, respectively. The morA gene encodes a short peptide, MorA, with an unusual initiation codon. Surprisingly, overexpression of the morA 5= untranslated region exhibited an inhibitory influence on colony formation of E. coli K-12.T he transition metal molybdenum is essential for life. In nature, molybdenum is present in various oxidation states and transported into organisms in the form of the tetraoxyanion molybdate. In the case of Escherichia coli, molybdate is transported through an ABC-type transport system encoded by the modABC operon (1). The expression of the modABC operon is repressed by the molybdate-bound ModE transcription factor (2). The ModE protein functions as a homodimer and consists of two domains, the N-terminal DNA-binding domain containing a winged helixturn-helix motif and the C-terminal molybdate-binding domain (3). Binding of molybdate to the C-terminal domain induces a conformational change of ModE so that it recognizes a palindromic sequence of its target promoters (4). In addition to repression of the molybdate transporter operon, the ModE-molybdate complex is involved in induction of three operons encoding molybdate-containing enzymes, dimethyl sulfoxide (DMSO) reductase (dmsABC), nitrate reductase (napFDAGHBC), and molybdenum cofactor synthase (moaABCDE) (5-7). The three known ModE-inducing targets are all involved in molybdenum metabolism and utilization. Since bacteria contain more than 50 species of the molybdate-containing enzyme, the repertoire of regulation targets of ModE could include more than the already-characterized operons.In this study, attempts were made to identify the set of regulation targets of the E. coli ModE transcription factor. For this purpose, we employed the genetic systematic evolution of ligands by exponential enrichment (SELEX) screening system, which was developed for identification of the set of regulation targets recognized by DNA-binding transcription factors and was successfully used for the search of regulation targets by AscG (8), AllR (9), CitB (10), Cra (11, 12), cyclic AMP receptor protein (CRP) (13), Dan (14), LeuO (15), NemA (16), PdhR (17), RcdA (18), PgrR (19), RstA (20), RutR (21), and TyrR (22). After the genomic SELEX screening, we identified at least 10 binding sites of the ModEmolybdate complex. Transcription in vivo of the predicted promoters located next to ...

Induction of the Escherichia coli yijE gene expression by cystine

“…The recombinant plasmids, pTW02 (yijE-lacZ), pHN01 (yihN-lacZ), and pDT04 (yhdT-lacZ), were confirmed by DNA sequencing using Lac30R primer. 13) Each lacZ translational fusion plasmid was transformed into MC4100, 14) and the transformant was infected with λ RS45 to prepare λ recombinant phage containing the lacZ fusion gene. E. coli BW25113 was infected with the λ lysate, and the lysogen containing the recombinant λ phage was selected by resistance to kanamycin.…”

The hierarchic network of metal-response transcription factors in Escherichia coli

“…93) In addition to repression of the molybdate transporter operon, the ModE-molybdate complex activates at six operons encoding molybdate-containing enzymes and three operons encoding as yet unidenfied genes. 94) Typical ModE-binding site is composed of an inverted repeat of nona-nucleotides, CGnTATATA, with a spacer of hexanucleotides. 94) 5.…”

“…94) Typical ModE-binding site is composed of an inverted repeat of nona-nucleotides, CGnTATATA, with a spacer of hexanucleotides. 94) 5. Cross-talk between the metal-sensing metal-response regulons…”

“…ModE directly activates six operons including four known molybdenum-dependent enzymes (modABCD, dsmABC, napFDAGHBC, and ynfEFGHI), which are induced under anaerobic condition in the dependent manner of FNR. 94,[98][99][100][101] ModE and the anaerobisis-sensing transcription factor FNR cooperatively function for activation of these promoters (Fig. 3(A)).…”

RaptRanker:in silicoRNA aptamer selection from HT-SELEX experiment based on local sequence and structure information

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