| Publications on this gene: |
1.
J Bacteriol 2006 Dec ; 23(188):8206-12.
In silico prediction and functional validation of sigma28-regulated genes in Chlamydia and Escherichia coli.
Yu HH ,Kibler D ,Tan M ,
Institute for Genomics and Bioinformatics, University of California, Irvine, California 92697-4025, USA.
sigma(28) RNA polymerase is an alternative RNA polymerase that has been proposed to have a role in late developmental gene regulation in Chlamydia, but only a single target gene has been identified. To discover additional sigma(28)-dependent genes in the Chlamydia trachomatis genome, we applied bioinformatic methods using a probability weight matrix based on known sigma(28) promoters in other bacteria and a second matrix based on a functional analysis of the sigma(28) promoter. We tested 16 candidate sigma(28) promoters predicted with these algorithms and found that 5 were active in a chlamydial sigma(28) in vitro transcription assay. hctB, the known sigma(28)-regulated gene, is only expressed late in the chlamydial developmental cycle only, and two of the newly identified sigma(28) target genes (tsp and tlyC_1) also have late expression profiles, providing support for sigma(28) as a regulator of late gene expression. One of the other novel sigma(28)-regulated genes is dnaK, a known heat shock-responsive gene, suggesting that sigma(28) RNA polymerase may be involved in the response to cellular stress. Our sigma(28) prediction algorithm can be applied to other bacteria, and by performing a similar analysis on the Escherichia coli genome, we have predicted and functionally identified five previously unknown sigma(28)-regulated genes in E. coli.
Publication Type: Research Support, N.I.H., Extramural;
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2.
Nucleic Acids Res 2006 ; 1(34):1-9.
Escherichia coli K-12: a cooperatively developed annotation snapshot--2005.
Riley M ,Abe T ,Arnaud MB ,Berlyn MK ,Blattner FR ,Chaudhuri RR ,Glasner JD ,Horiuchi T ,Keseler IM ,Kosuge T ,Mori H ,Perna NT ,Plunkett G 3rd,Rudd KE ,Serres MH ,Thomas GH ,Thomson NR ,Wishart D ,Wanner BL ,
Josephine Bay Paul Center, Marine Biological Laboratory, Woods Hole, MA 02543, USA. mriley@mbl.edu
The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.;
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3.
Nucleic Acids Res 2006 Jan 1; Database issue(34):D181-6.
TCDB: the Transporter Classification Database for membrane transport protein analyses and information.
Saier MH Jr,Tran CV ,Barabote RD ,
Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA. msaier@ucsd.edu
The Transporter Classification Database (TCDB) is a web accessible, curated, relational database containing sequence, classification, structural, functional and evolutionary information about transport systems from a variety of living organisms. TCDB is a curated repository for factual information compiled from >10,000 references, encompassing approximately 3000 representative transporters and putative transporters, classified into >400 families. The transporter classification (TC) system is an International Union of Biochemistry and Molecular Biology approved system of nomenclature for transport protein classification. TCDB is freely accessible at http://www.tcdb.org. The web interface provides several different methods for accessing the data, including step-by-step access to hierarchical classification, direct search by sequence or TC number and full-text searching. The functional ontology that underlies the database structure facilitates powerful query searches that yield valuable data in a quick and easy way. The TCDB website also offers several tools specifically designed for analyzing the unique characteristics of transport proteins. TCDB not only provides curated information and a tool for classifying newly identified membrane proteins, but also serves as a genome transporter-annotation tool.
Publication Type: Research Support, N.I.H., Extramural;
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4.
J Am Chem Soc 2004 Aug 25; 33(126):10296-305.
Oxyanion selectivity in sulfate and molybdate transport proteins: an ab initio/CDM study.
Dudev T ,Lim C ,
Contribution from the Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, R.O.C.
A striking feature of sulfate (SO(4)(2-)) and molybdate (MoO(4)(2-)) transport proteins, such as SBP and ModA, which specifically bind SO(4)(2-) and MoO(4)(2-), respectively, is their ability to discriminate very similar anions with the same net charge, geometry, and hydrogen-bonding properties. Here, we determine to what extent (1) oxyanion-solvent interactions, (2) oxyanion-amino acid interactions, and (3) the anion-binding pocket sizes of the cognate protein contribute to the anion selectivity process in SO(4)(2-) and MoO(4)(2-) transport proteins by computing the free energies for replacing SO(4)(2-) with MoO(4)(2)(-)/WO(4)(2-) in model SO(4)(2-)-binding sites of varying degrees of solvent exposure using a combined quantum mechanical/continuum dielectric approach. The calculations reveal that MoO(4)(2-) transport proteins, such as ModA, specifically bind MoO(4)(2-)/WO(4)(2-) but not SO(4)(2-), mainly because the desolvation penalty of MoO(4)(2-)/WO(4)(2-) is significantly less than that of SO(4)(2-) and, to a lesser extent, because the large and rigid cavity in these proteins attenuates ligand interactions with SO(4)(2-), as compared to MoO(4)(2-). On the other hand, SO(4)(2-) transport proteins prefer SO(4)(2-) to MoO(4)(2-)/WO(4)(2-) because the small anion-binding pocket characteristic of these proteins inhibits binding of the larger MoO(4)(2-) and WO(4)(2-) anions. The calculations also help to explain the absence of positively charged Lys/Arg side chains in the anion-binding sites of SBP and ModA. During evolution, these transport proteins may have excluded cationic ligands from their binding sites because, on one hand, Lys/Arg do not contribute to the selectivity of the binding pocket and, on the other, they substantially stabilize the complex between the oxyanion and protein ligands, which in turn would prohibit the rapid release of the bound oxyanion at a certain stage during the transport process.
Publication Type: Research Support, Non-U.S. Gov't;
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5.
FEMS Microbiol Lett 2003 Jan 21; 1(218):187-93.
In vivo detection of molybdate-binding proteins using a competition assay with ModE in Escherichia coli.
Kuper J ,Meyer zu Berstenhorst S ,Vödisch B ,Mendel RR ,Schwarz G ,Boxer DH ,
Department of Plant Biology, Technical University of Braunschweig, 38023, Braunschweig, Germany.
Molybdenum is an important trace element as it forms the essential part of the active site in all molybdenum-containing enzymes. We have designed an assay for the in vivo detection of molybdate binding to proteins in Escherichia coli. The assay is based on (i). the molybdate-dependent transcriptional regulation of the moa operon by the ModE protein, and (ii). the competition for molybdate between ModE and other molybdate-binding proteins in the cytoplasm of E. coli. We were able to verify in vivo molybdate binding to three different bacterial proteins that are known to bind molybdate. This sensitive in vivo system allows the testing of different proteins for molybdate binding under in vivo conditions and will facilitate the identification of other cellular factors needed for molybdate binding. As a first example, we examined the eukaryotic protein Cnx1 that is involved in the last step of molybdenum cofactor biosynthesis in plants, and show that it is able to compete with ModE for molybdate in a molybdopterin-dependent fashion.
Publication Type: Research Support, Non-U.S. Gov't;
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6.
J Biol Chem 1999 Aug 20; 34(274):24308-15.
An analysis of the binding of repressor protein ModE to modABCD (molybdate transport) operator/promoter DNA of Escherichia coli.
Grunden AM ,Self WT ,Villain M ,Blalock JE ,Shanmugam KT ,
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611, USA.
Expression of the modABCD operon in Escherichia coli, which codes for a molybdate-specific transporter, is repressed by ModE in vivo in a molybdate-dependent fashion. In vitro DNase I-footprinting experiments identified three distinct regions of protection by ModE-molybdate on the modA operator/promoter DNA, GTTATATT (-15 to -8; region 1), GCCTACAT (-4 to +4; region 2), and GTTACAT (+8 to +14; region 3). Within the three regions of the protected DNA, a pentamer sequence, TAYAT (Y = C or T), can be identified. DNA-electrophoretic mobility experiments showed that the protected regions 1 and 2 are essential for binding of ModE-molybdate to DNA, whereas the protected region 3 increases the affinity of the DNA to the repressor. The stoichiometry of this interaction was found to be two ModE-molybdate per modA operator DNA. ModE-molybdate at 5 nM completely protected the modABCD operator/promoter DNA from DNase I-catalyzed hydrolysis, whereas ModE alone failed to protect the DNA even at 100 nM. The apparent K(d) for the interaction between the modA operator DNA and ModE-molybdate was 0.3 nM, and the K(d) increased to 8 nM in the absence of molybdate. Among the various oxyanions tested, only tungstate replaced molybdate in the repression of modA by ModE, but the affinity of ModE-tungstate for modABCD operator DNA was 6 times lower than with ModE-molybdate. A mutant ModE(T125I) protein, which repressed modA-lac even in the absence of molybdate, protected the same region of modA operator DNA in the absence of molybdate. The apparent K(d) for the interaction between modA operator DNA and ModE(T125I) was 3 nM in the presence of molybdate and 4 nM without molybdate. The binding of molybdate to ModE resulted in a decrease in fluorescence emission, indicating a conformational change of the protein upon molybdate binding. The fluorescence emission spectra of mutant ModE proteins, ModE(T125I) and ModE(Q216*), were unaffected by molybdate. The molybdate-independent mutant ModE proteins apparently mimic in its conformation the native ModE-molybdate complex, which binds to a DNA sequence motif of TATAT-7bp-TAYAT.
Publication Type: Research Support, U.S. Gov't, P.H.S.;
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7.
Biochim Biophys Acta 1998 Mar 13; 2(1370):337-46.
Molybdate binding by ModA, the periplasmic component of the Escherichia coli mod molybdate transport system.
Imperial J ,Hadi M ,Amy NK ,
Department of Nutritional Sciences, University of California-Berkeley, Berkeley, CA 94720, USA. jimperial@bit.etsia.upm.es
ModA, the periplasmic-binding protein of the Escherichia coli mod transport system was overexpressed and purified. Binding of molybdate and tungstate to ModA was found to modify the UV absorption and fluorescence emission spectra of the protein. Titration of these changes showed that ModA binds molybdate and tungstate in a 1:1 molar ratio. ModA showed an intrinsic fluorescence emission spectrum attributable to its three tryptophanyl residues. Molybdate binding caused a conformational change in the protein characterized by: (i) a shift of tryptophanyl groups to a more hydrophobic environment; (ii) a quenching (at pH 5.0) or enhancement (at pH 7.8) of fluorescence; and (iii) a higher availability of tryptophanyl groups to the polar quencher acrylamide. The tight binding of molybdate did not allow an accurate estimation of the binding constants by these indirect methods. An isotopic binding method with 99MoO42- was used for accurate determination of KD (20 nM) and stoichiometry (1:1 molar ratio). ModA bound tungstate with approximately the same affinity, but did not bind sulfate or phosphate. These KDs are 150- to 250-fold lower than those previously reported, and compatible with the high molybdate transport affinity of the mod system. The affinity of ModA for molybdate was also determined in vivo and found to be similar to that determined in vitro.
Publication Type: Research Support, Non-U.S. Gov't;
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8.
Arch Microbiol 1997 Nov ; 5(168):345-54.
Molybdate transport and regulation in bacteria.
Grunden AM ,Shanmugam KT ,
Department of Microbiology and Cell Science, 110700, University of Florida, Gainesville, FL 32611-0700, USA.
Molybdate is transported in bacteria by a high-affinity transport system composed of a periplasmic binding protein, an integral membrane protein, and an energizer protein. These three proteins are coded by modA, modB, and modC genes, respectively. The ModA, ModB, and ModC proteins from various organisms (Escherichia coli, Haemophilus influenzae, Azotobacter vinelandii, and Rhodobacter capsulatus) are very similar. The lowest Km value reported for molybdate in the molybdate transport process is approximately 50 nM. In a mod mutant, molybdate is transported by the sulfate transport system or by a nonspecific anion transporter. Molybdate transport is tightly coupled to utilization in E. coli and Klebsiella pneumoniae, while other dinitrogen-fixing organisms appear to have a molybdenum storage protein. In all organisms studied so far, molybdate transport genes are regulated by a repressor protein, ModE. The ModE-molybdate complex binds to the sequences TAYAT (Y = T or C) in the operator/ promoter region in E. coli and prevents transcription of the modABCD operon. The ModE-molybdate complex binds to DNA as a homodimer in E. coli and possibly in other organisms as well. In R. capsulatus, however, two ModE homologues (MopAB proteins) are required for repression.
Publication Type: Research Support, U.S. Gov't, P.H.S.; Review;
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9.
Nat Struct Biol 1997 Sep ; 9(4):703-7.
Crystal structure of the molybdate binding protein ModA.
Hu Y ,Rech S ,Gunsalus RP ,Rees DC ,
Publication Type: Letter; Research Support, U.S. Gov't, P.H.S.;
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11.
Mol Microbiol 1997 Feb ; 3(23):515-24.
Characterization of the ModE DNA-binding sites in the control regions of modABCD and moaABCDE of Escherichia coli.
McNicholas PM ,Rech SA ,Gunsalus RP ,
Department of Microbiology and Molecular Genetics, University of California at Los Angeles 90095-1489, USA.
The Escherichia coli molybdate transporter, encoded by the modABCD operon, is negatively regulated by the modE gene product in response to the intracellular molybdate concentration. Utilizing an in vivo titration assay, we localized the ModE-binding site to the start of modA transcription. This localization was further characterized using in vitro gel-shift assays and DNase I footprinting. ModE bound the wild-type modA promoter with an apparent dissociation constant (Kd) of 45 nM, and addition of molybdate, in physiologically relevant amounts, significantly increased DNA binding. Consistent with these data, modA promoter fragments containing mutations that reduced ModE repression in vivo displayed proportionately higher apparent Kd values in vitro. DNase I footprinting of the modA promoter revealed a single protected region that overlapped the start site of transcription and extended from position -18 to +10, relative to the transcript start site. Gel-shifting assays, employing the promoter regions from the tor, nrf, moa and moe operons, revealed that ModE bound only the moa promoter region, with an apparent Kd of 24nM. Footprint analysis of the moaA promoter revealed a single protected region located immediately upstream of the putative -35 consensus sequence and extending from position -202 to -174, relative to the start of translation. In vivo expression of a moaA-lacZ operon fusion was stimulated twofold by ModE. However, relative to modA, binding of ModE to the moaA promoter appeared to be largely molybdate independent both in vitro and in vivo. These findings demonstrate that ModE acts both as a repressor and activator of the mod and moa operons, respectively, depending on the properties of the binding site.
Publication Type: Research Support, U.S. Gov't, P.H.S.;
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12.
FEMS Microbiol Lett 1996 Nov 15; 1(145):117-23.
The Escherichia coli modE gene: effect of modE mutations on molybdate dependent modA expression.
McNicholas PM ,Chiang RC ,Gunsalus RP ,
Department of Microbiology and Molecular Genetics, University of California at Los Angeles 90095-1489, USA.
The Escherichia coli modABCD operon, which encodes a high-affinity molybdate uptake system, is transcriptionally regulated in response to molybdate availability by ModE. Here we describe a highly effective enrichment protocol, applicable to any gene with a repressor role, and establish its application in the isolation of transposon mutations in modE. In addition we show that disruption of the ModE C-terminus abolishes derepression in the absence of molybdate, implying this region of ModE controls the repressor activity. Finally, a mutational analysis of a proposed molybdate binding motif indicates that this motif does not function in regulating the repressor activity of ModE.
Publication Type: Research Support, U.S. Gov't, P.H.S.;
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14.
Nucleic Acids Symp Ser 1995 ; 34():91-2.
Characterization and transcriptional regulation of the modABCD genes for molybdenum transport in Escherichia coli.
Miyake H ,Yabu H ,Satoh T ,Yamamoto I ,
Department of Biological Sciences, Faculty of Science, Hiroshima University, Japan.
The mod genes coding for molybdenum transport system were isolated from Escherichia coli by means of complementation of the tor mutation that causes no synthesis of some molybdenum-containing enzymes. The nucleotide sequence of 3048-base pair showed four open reading frames including the previously reported modC gene. The genes were named modA, modB, modC, and modD. Promoter analysis by lacZ assay and primer extension showed the genes modA and modB individually have their own promoters. No transcriptional regulation of the two genes were observed in the presence of molybdate, nitrate, and oxygen in E. coli strain DH5 alpha.
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15.
Biochimie 1995 ; 9(77):744-50.
A family of homologous substrate-binding proteins with a broad range of substrate specificity and dissimilar biological functions.
Wu LF ,Mandrand-Berthelot MA ,
Institut National des Sciences Appliquées, Villeurbanne, France.
The uptake of peptides is accomplished mainly by a family of homologous oligopeptide or dipeptide transporters in bacteria. Computer-aided sequence analyses expand members of the oligopeptide-binding protein family to nickel and heme permeases and other proteins, including an enzyme hyaluronate synthase. They are involved in human pathogenicity, bacterial virulence, substrate-sensing, bacterial conjugation and bacterial metabolic reactions distinct from nutrient uptake. These homologous proteins are found in both purple bacteria and Gram-positive bacteria, indicating the presence of a common ancestor before the appearance of the two eubacterial phyla. Nevertheless, the pheromone-binding proteins, involved in bacterial conjugation, and the hyaluronate synthase are present only in the low G-C Gram-positive eubacteria subdivision, which suggests that these proteins diverged from the common ancestor after the appearance of this subdivision.
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16.
J Bacteriol 1996 Jul ; 14(178):4310-2.
Molybdenum cofactor biosynthesis in Escherichia coli mod and mog mutants.
Joshi MS ,Johnson JL ,Rajagopalan KV ,
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
The molybdopterin content of Escherichia coli mod and mog mutants was estimated by conversion to the form A derivative. The results are in accord with complete phenotypic repair of mod, and incomplete repair of mog, by culture in high concentrations of molybdate. A possible role for Mog as a molybdochelatase is discussed.
Publication Type: Research Support, U.S. Gov't, P.H.S.;
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17.
J Biol Chem 1996 Feb 2; 5(271):2557-62.
Properties of the periplasmic ModA molybdate-binding protein of Escherichia coli.
Rech S ,Wolin C ,Gunsalus RP ,
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90095, USA.
The modABCD operon, located at 17 min on the Escherichia coli chromosome, encodes the protein components of a high affinity molybdate uptake system. Sequence analysis of the modA gene (GenBank L34009) predicts that it encodes a periplasmic binding protein based on the presence of a leader-like sequence at its N terminus. To examine the properties of the ModA protein, the modA structural gene was overexpressed, and its product was purified. The ModA protein was localized to the periplasmic space of the cell, and it was released following a gentle osmotic shock. The N-terminal sequence of ModA confirmed that a leader region of 24 amino acids was removed upon export from the cell. The apparent size of ModA is 31.6 kDa as determined by gel sieve chromatography, whereas it is 22.5 kDa when examined by SDS-polyacrylamide gel electrophoresis. A ligand-dependent protein mobility shift assay was devised using a native polyacrylamide gel electrophoresis protocol to examine binding of molybdate and other anions to the ModA periplasmic protein. Whereas molybdate and tungstate were bound with high affinity (approximately 5 microM), sulfate, chromate, selenate, phosphate, and chlorate did not bind even when tested at 2 mM. A UV spectral assay revealed apparent Kd values of binding for molybdate and tungstate of 3 and 7 microM, respectively. Strains defective in the modA gene were unable to transport molybdate unless high levels of the anion were supplied in the medium. Therefore the modA gene product is essential for high affinity molybdate uptake by the cell. Tungstate interference of molybdate acquisition by the cell is apparently due in part to the high affinity of the ModA protein for this anion.
Publication Type: Research Support, U.S. Gov't, P.H.S.;
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18.
Microbiol Res 1995 Nov ; 4(150):347-61.
Molecular analysis of the molybdate uptake operon, modABCD, of Escherichia coli and modR, a regulatory gene.
Walkenhorst HM ,Hemschemeier SK ,Eichenlaub R ,
Lehrstuhl Gentechnologie/Mikrobiologie, Fakultät für Biologie, Universität Bielefeld, Germany.
The nucleotide sequence of a 6.8-kb chromosomal subfragment of plasmid pHW100 complementing an Escherichia coli modC (chlD) mutant has been determined. This DNA region encodes the genes of a high-affinity uptake system for molybdate arranged in an operon with the genes modABCD. Since the modA product has a signal peptide at the N-terminus it probably is the periplasmic binding-protein for molybdate. The products of modB (chlJ) and modC (chlD) have been described earlier as the inner membrane protein and the ATP-binding protein of the molybdate transport system, respectively. At present, there is no information on possible functions of the fourth gene of the operon, modD. Upstream of the mod operon, two other gene loci, termed modR and an open reading frame ORF6 could be identified. ModR shares homology with a molybdenum-pterin binding protein of Clostridium pasteurianum. ORF6 has extensive homology to ModC and other nucleotide-binding proteins of E. coli. Insertional inactivation of modR and ORF6 using a gentamicin resistance gene cartridge has no effect on molybdoenzyme activities, indicating that none of the two gene products is essential for molybdate uptake or molybdenum cofactor synthesis. However, by using a plasmid carrying a modA-lacZ gene fusion we observed that inactivation of modR releases repression of the mod operon independent of the molybdate concentration in the medium. This indicates that modR is a component of the mod operon regulation or the repressor itself.
Publication Type: Research Support, Non-U.S. Gov't;
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19.
J Bacteriol 1996 Feb ; 3(178):735-44.
Repression of the Escherichia coli modABCD (molybdate transport) operon by ModE.
Grunden AM ,Ray RM ,Rosentel JK ,Healy FG ,Shanmugam KT ,
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA.
The modABC gene products constitute the molybdate-specific transport system in Escherichia coli. Another operon coding for two proteins which diverges from the modABCD operon has been identified. The first gene of this operon codes for a 262-amino-acid protein, designated ModE (28 kDa), and the second genes codes for a 490-amino-acid protein. ModF (54 kDa). The role of ModF has not yet been determined; however, mutations in modE depressed modABCD transcription even in the presence of molybdate, suggesting that ModE is a repressor. ModE, in the presence of 1 mM molybdate, repressed the production of plasmid-encoded ModA and ModB' proteins in an in vitro transcription-translation system. DNA mobility shift experiments confirmed that ModE binds to an oligonucleotide derived from the operator region of the modABCD operon. Further experimentation indicated that ModE binding to target DNA minimally requires an 8-bp inverted-repeat sequence, TAAC GITA. A highly conserved amino acid sequence, TSARNOXXG (amino acids 125 to 133), was identified in ModE and homologs from Azotobacter vinelandii, Haemophilus influenzae, Rhodobacter capsulatus, and Clostridium pasterianum. Mutants with mutations in either T or G of this amino acid sequence were isolated as "superrepressor" mutants. These mutant proteins repressed modABCD transcription even in the absence of molybdate, which implies that this stretch of amino acids is essential for the binding of molybdate by the ModE protein. These results show that molybdate transport in E. coli is regulated by ModE, which acts as a repressor when bound to molybdate.
Publication Type: Research Support, U.S. Gov't, P.H.S.;
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20.
J Bacteriol 1995 Feb ; 4(177):1023-9.
Regulation of the molybdate transport operon, modABCD, of Escherichia coli in response to molybdate availability.
Rech S ,Deppenmeier U ,Gunsalus RP ,
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.
The mod (chlD) locus at 17 min on the Escherichia coli chromosome encodes a high-affinity molybdate uptake system. To further investigate the structure and regulation of these genes, the DNA region upstream of the previously identified modBC (chlJD) genes was cloned and sequenced. A single open reading frame, designated modA, was identified and appears to encode a periplasmic binding protein for the molybdate uptake system. To determine how the mod genes are regulated in response to molybdate, nitrate, and oxygen, we constructed a series of mod-lacZ operon fusions to the upstream region and introduced them in single copy onto the E. coli chromosome. Whereas molybdate limitation resulted in elevated mod-lacZ expression, neither oxygen nor nitrate had any significant effect on gene expression. A regulatory motif, CATAA, located at the modA promoter was identified and shown to be required for molybdate-dependent control of the modABCD operon. Mutations within this sequence resulted in nearly complete derepression of gene expression and suggest that transcription of the operon is mediated by a molybdenum-responsive regulatory protein.
Publication Type: Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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21.
J Bacteriol 1995 Sep ; 17(177):4857-64.
Molybdate and regulation of mod (molybdate transport), fdhF, and hyc (formate hydrogenlyase) operons in Escherichia coli.
Rosentel JK ,Healy F ,Maupin-Furlow JA ,Lee JH ,Shanmugam KT ,
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA.
Escherichia coli mutants with defined mutations in specific mod genes that affect molybdate transport were isolated and analyzed for the effects of particular mutations on the regulation of the mod operon as well as the fdhF and hyc operons which code for the components of the formate hydrogenlyase (FHL) complex. phi (hyc'-'lacZ+) mod double mutants produced beta-galactosidase activity only when they were cultured in medium supplemented with molybdate. This requirement was specific for molybdate and was independent of the moa, mob, and moe gene products needed for molybdopterin guanine dinucleotide (MGD) synthesis, as well as Mog protein. The concentration of molybdate required for FHL production by mod mutants was dependent on medium composition. In low-sulfur medium, the amount of molybdate needed by mod mutants for the production of half-maximal FHL activity was increased approximately 20 times by the addition of 40 mM of sulfate, mod mutants growing in low-sulfur medium transported molybdate through the sulfate transport system, as seen by the requirement of the cysA gene product for this transport. In wild-type E. coli, the mod operon is expressed at very low levels, and a mod+ merodiploid E. coli carrying a modA-lacZ fusion produced less than 20 units of beta-galactosidase activity. This level was increased by over 175 times by a mutation in the modA, modB, or modC gene. The addition of molybdate to the growth medium of a mod mutant lowered phi (modA'-'lacZ+) expression. Repression of the mod operon was sensitive to molybdate but was insensitive to mutations in the MGD synthetic pathway. These physiological and genetic experiments show that molybdate can be transported by one of the following three anion transport system in E. coli: the native system, the sulfate transport system (cysTWA gene products), and an undefined transporter. Upon entering the cytoplasm, molybdate branches out to mod regulation, fdhF and hyc activation, and metabolic conversion, leading to MGD synthesis and active molybdoenzyme synthesis.
Publication Type: Research Support, U.S. Gov't, P.H.S.;
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22.
J Bacteriol 1995 Sep ; 17(177):4851-6.
Genetic analysis of the modABCD (molybdate transport) operon of Escherichia coli.
Maupin-Furlow JA ,Rosentel JK ,Lee JH ,Deppenmeier U ,Gunsalus RP ,Shanmugam KT ,
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA.
DNA sequence analysis of the modABCD operon of Escherichia coli revealed the presence of four open reading frames. The first gene, modA, codes for a 257-amino-acid periplasmic binding protein enunciated by the presence of a signal peptide-like sequence. The second gene (modB) encodes a 229-amino-acid protein with a potential membrane location, while the 352-amino-acid ModC protein (modC product) contains a nucleotide-binding motif. On the basis of sequence similarities with proteins from other transport systems and molybdate transport proteins from other organisms, these three proteins are proposed to constitute the molybdate transport system. The fourth open reading frame (modD) encodes a 231-amino-acid protein of unknown function. Plasmids containing different mod genes were used to map several molybdate-suppressible chlorate-resistant mutants; interestingly, none of the 40 mutants tested had a mutation in the modD gene. About 35% of these chlorate-resistant mutants were not complemented by mod operon DNA. These mutants, designated mol, contained mutations at unknown chromosomal location(s) and produced formate hydrogenlyase activity only when cultured in molybdate-supplemented glucose-minimal medium, not in L broth. This group of mol mutants constitutes a new class of molybdate utilization mutants distinct from other known mutants in molybdate metabolism. These results show that molybdate, after transport into cells by the ModABC proteins, is metabolized (activated?) by the products of the mol gene(s).
Publication Type: Research Support, U.S. Gov't, P.H.S.;
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