Regulatory effects of potassium and inorganic anions on the NADP-specific malic enzyme of Escherichia coli

The effects of K+ and various anions on the catalytic and regulatory properties of the NADP-specific malic enzyme of Escherichia coli are reported. Studies on the susceptibility of the enzyme to proteolysis indicate that K+ binds directly to the enzyme with a resultant change in enzyme conformation. Kinetic studies indicate that the binding of optimal concentrations of K+ results in activation of the enzyme, increasing both the Vmax and the affinity of the enzyme for divalent cations. The inhibition of enzyme activity observed at KCl concentrations greater than 50 mM is shown to be nonspecific, resulting from increasing ionic strength. The mixed cooperativity between malate-binding sites previously reported at optimal K+ concentration is more pronounced at nonoptimal K+ concentrations (0 and 150 mM). The regulatory effect of metal cofactors and the mixed cooperativity between malate-binding sites is abolished when kinetic studies are conducted at low ionic strength or in the presence of acetate. Acetate appears to act as an activator, increasing the affinity of the enzyme for malate and protecting the enzyme against the inhibition caused by high ionic strength. It is postulated that the enzyme is operating in vivo in a partially inhibited state owing to the ionic strength of the cytoplasm. The kinetic studies conducted at higher ionic strength in vitro are therefore more applicable to the in vivo situation.

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CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from Enterotoxigenic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00131-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5060-5067 ◽

Cited By ~ 33

Author(s):

M. Carolina Pilonieta ◽

Maria D. Bodero ◽

George P. Munson

Keyword(s):

Escherichia Coli ◽

Binding Sites ◽

Secretory Pathway ◽

Dnase I ◽

Enterotoxigenic Escherichia Coli ◽

Watery Diarrhea ◽

Dnase I Footprinting ◽

Virulence Regulator

ABSTRACT H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.

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Binding and transcriptional activation of non-flagellar genes by the Escherichia coli flagellar master regulator FlhD2C2

Microbiology ◽

10.1099/mic.0.27879-0 ◽

2005 ◽

Vol 151 (6) ◽

pp. 1779-1788 ◽

Cited By ~ 46

Author(s):

Graham P. Stafford ◽

Tomoo Ogi ◽

Colin Hughes

Keyword(s):

Escherichia Coli ◽

Dna Sequences ◽

Transcriptional Activation ◽

Binding Sites ◽

Class Ii ◽

Promoter Regions ◽

E Coli ◽

Flagellar Genes

The gene hierarchy directing biogenesis of peritrichous flagella on the surface of Escherichia coli and other enterobacteria is controlled by the heterotetrameric master transcriptional regulator FlhD2C2. To assess the extent to which FlhD2C2 directly activates promoters of a wider regulon, a computational screen of the E. coli genome was used to search for gene-proximal DNA sequences similar to the 42–44 bp inverted repeat FlhD2C2 binding consensus. This identified the binding sequences upstream of all eight flagella class II operons, and also putative novel FlhD2C2 binding sites in the promoter regions of 39 non-flagellar genes. Nine representative non-flagellar promoter regions were all bound in vitro by active reconstituted FlhD2C2 over the K D range 38–356 nM, and of the nine corresponding chromosomal promoter–lacZ fusions, those of the four genes b1904, b2446, wzz fepE and gltI showed up to 50-fold dependence on FlhD2C2 in vivo. In comparison, four representative flagella class II promoters bound FlhD2C2 in the K D range 12–43 nM and were upregulated in vivo 30- to 990-fold. The FlhD2C2-binding sites of the four regulated non-flagellar genes overlap by 1 or 2 bp the predicted −35 motif of the FlhD2C2-activated σ 70 promoters, as is the case with FlhD2C2-dependent class II flagellar promoters. The data indicate a wider FlhD2C2 regulon, in which non-flagellar genes are bound and activated directly, albeit less strongly, by the same mechanism as that regulating the flagella gene hierarchy.

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Development of muscimol binding sites in chick embryo neural retina in vivo and in vitro: Regulatory effects of cyclic AMP

International Journal of Developmental Neuroscience ◽

10.1016/0736-5748(85)90040-1 ◽

1985 ◽

Vol 3 (5) ◽

pp. 511-515 ◽

Cited By ~ 6

Author(s):

Paul Madtes ◽

Ruben Adler

Keyword(s):

Chick Embryo ◽

Cyclic Amp ◽

Binding Sites ◽

Neural Retina ◽

Regulatory Effects ◽

Muscimol Binding

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Enhancement of [3H]DAGO1 binding to rat brain by low concentrations of monovalent cations

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y87-370 ◽

1987 ◽

Vol 65 (11) ◽

pp. 2338-2345 ◽

Cited By ~ 3

Author(s):

Gordon T. Bolger ◽

Kendall A. Marcus ◽

Ronald Thibou ◽

Phil Skolnick ◽

Ben Avi Weissman

Keyword(s):

Rat Brain ◽

Binding Sites ◽

Divalent Cations ◽

Opiate Receptors ◽

Opiate Receptor ◽

Affinity Binding ◽

High Affinity ◽

Regulatory Effects ◽

In Vitro Effects

The effects of mono- and di-valent cations and the nonhydrolyzable guanyl nucleotide derivative 5′-guanylimidodiphosphate (Gpp(NH)p) on the binding of the selective, high affinity μ-opiate receptor agonist, [3H]DAGO ([3H]Tyr-D-Ala-Gly-Mephe-Gly-ol), to rat brain membranes were studied in a low ionic strength 5 mM Tris–HCl buffer. Na+ and Li+ (50 mM) maximally increased [3H]DAGO binding (EC50 values for Na+,2.9 mM and Li, 6.2 mM) by revealing a population of low affinity binding sites. The density of high affinity [3H]DAGO binding sites was unaffected by Na+ and Li+, but was maximally increased by 50 mM K+ and Rb+ (EC50 values for K+, 8.5 mM and Rb+, 12.9 mM). Divalent cations (Ca2+, Mg2+; 50 mM) inhibited [3H]DAGO binding. Gpp(NH)p decreased the affinity of [3H]DAGO binding, an effect that was enhanced by Na+ but not by K+. The binding of the μ-agonist [3H]dihydromorphine was unaffected by 50 mM Na+ in 5 mM Tris–HCl. In 50 mM Tris–HCl, Na+ (50 mM) inhibited [3H]DAGO binding by decreasing the density of high affinity binding sites and promoting low affinity binding. The effects of Na+ in 5 mM and 50 mM Tris–HCl were also investigated on the binding of other opiate receptor agonists and antagonists. [3H]D-Ala-D-Leu-enkephalin binding was increased and inhibited, [3H]etorphine binding increased and was unchanged, and both [3H]bremazocine and [3H]naloxone binding increased by 50 mM Na+ in 5 mM and 50 mM Tris–HCl, respectively. These findings indicate that the in vitro effects of Na+ at μ- and possibly other opiate receptors in rat brain are dependent on the concentration of Tris–HCl used in the assay buffer, lower concentrations of Tris-HCl revealing novel regulatory effects for Na+ at μ-opiate receptors.

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Single-Target Regulators Constitute the Minority Group of Transcription Factors in Escherichia coli K-12

Frontiers in Microbiology ◽

10.3389/fmicb.2021.697803 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tomohiro Shimada ◽

Hiroshi Ogasawara ◽

Ikki Kobayashi ◽

Naoki Kobayashi ◽

Akira Ishihama

Keyword(s):

Escherichia Coli ◽

Transcription Factors ◽

Binding Sites ◽

Target Genes ◽

Minority Group ◽

E Coli ◽

Single Target ◽

K 12

The identification of regulatory targets of all transcription factors (TFs) is critical for understanding the entire network of genome regulation. A total of approximately 300 TFs exist in the model prokaryote Escherichia coli K-12, but the identification of whole sets of their direct targets is impossible with use of in vivo approaches. For this end, the most direct and quick approach is to identify the TF-binding sites in vitro on the genome. We then developed and utilized the gSELEX screening system in vitro for identification of more than 150 E. coli TF-binding sites along the E. coli genome. Based on the number of predicted regulatory targets, we classified E. coli K-12 TFs into four groups, altogether forming a hierarchy ranging from a single-target TF (ST-TF) to local TFs, global TFs, and nucleoid-associated TFs controlling as many as 1,000 targets. Using the collection of purified TFs and a library of genome DNA segments from a single and the same E. coli K-12, we identified here a total of 11 novel ST-TFs, CsqR, CusR, HprR, NorR, PepA, PutA, QseA, RspR, UvrY, ZraR, and YqhC. The regulation of single-target promoters was analyzed in details for the hitherto uncharacterized QseA and RspR. In most cases, the ST-TF gene and its regulatory target genes are adjacently located on the E. coli K-12 genome, implying their simultaneous transfer in the course of genome evolution. The newly identified 11 ST-TFs and the total of 13 hitherto identified altogether constitute the minority group of TFs in E. coli K-12.

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Microbial metabolism of amino alcohols. Formation of coenzyme B12-dependent ethanolamine ammonia-lyase and its concerted induction in Escherichia coli

Biochemical Journal ◽

10.1042/bj1760751 ◽

1978 ◽

Vol 176 (3) ◽

pp. 751-757 ◽

Cited By ~ 16

Author(s):

C M Blackwell ◽

J M Turner

Keyword(s):

Escherichia Coli ◽

Enzyme Stability ◽

Kinetic Studies ◽

Coenzyme B12 ◽

E Coli ◽

Immunological Tests ◽

Enzyme Formation ◽

Acting In Concert

1. Kinetic studies of ethanolamine ammonia-lyase formation by Escherichia coli suggested that coenzyme B12 (5′-deoxyadenosylcobalamin), with ethanolamine, is a co-inducer. 2. Enzymic and immunological tests failed to show the formation of complementary enzyme components induced separately by ethanolamine and cobalamin respectively. 3. Although specific for ethanolamine as the substrate, enzyme formation was induced by certain analogues, e.g. 2-aminopropan-1-ol. 4. Experiments with cyano[57Co]-cobalamin suggested that neither coenzyme B12 nor some more tightly bound coenzymically inactive cobamide was necessary for enzyme stability in vitro. 5. Mutants of E. coli were obtained which formed ethanolamine ammonia-lyase apoenzyme constitutively, showing that neither ethanolamine nor cobalamin was required for assembly or post-transcriptional stability of the enzyme in vivo. Constitutive enzyme formation was subject to catabolite repression, particularly by glucose. 6. It appears likely that ethanolamine and coenzyme B12, acting in concert, induce ethanolamine ammonia-lyase formation. The term ‘concerted’ induction is proposed for this phenomenon.

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Regulation of extracellular matrix assembly: in vitro reconstitution of a partial fertilization envelope from isolated components.

The Journal of Cell Biology ◽

10.1083/jcb.105.1.561 ◽

1987 ◽

Vol 105 (1) ◽

pp. 561-567 ◽

Cited By ~ 16

Author(s):

P J Weidman ◽

B M Shapiro

Keyword(s):

Binding Sites ◽

Divalent Cations ◽

Matrix Assembly ◽

Sea Urchin Egg ◽

In Vitro Reconstitution ◽

Cross Links ◽

Necessary And Sufficient ◽

Secreted Peptides

At fertilization, the glycocalyx (vitelline layer) of the sea urchin egg is transformed into an elevated fertilization envelope by the association of secreted peptides and the formation of intermolecular dityrosine bonds. Dityrosine cross-links are formed by a secreted ovoperoxidase that exists in a Ca2+-stabilized complex with proteoliaisin in the fertilization envelope. By using purified proteins, we now show that proteoliaisin is necessary and sufficient to link ovoperoxidase to the egg glycocalyx. Specifically, we have found that ovoperoxidase can associate with the vitelline layer only when complexed with proteoliaisin; proteoliaisin binds to the vitelline layer independently of its association with ovoperoxidase; proteolytic modification of the vitelline layer is not required for this interaction to occur; the binding of proteoliaisin to the vitelline layer is mediated by the synergistic action of the two major seawater divalent cations, Ca2+ and Mg2+; the number of proteoliaisin-binding sites on the vitelline layer of unfertilized eggs is equivalent to the amount of proteoliaisin secreted at fertilization; and the binding of ovoperoxidase to the vitelline layer, via proteoliaisin, permits the in vitro cross-linking of these two in vivo substrates. The association of purified ovoperoxidase and proteoliaisin with the vitelline layer of unfertilized eggs reconstitutes part of the morphogenesis of the fertilization envelope.

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Role of RepA and DnaA Proteins in the Opening of the Origin of DNA Replication of an IncB Plasmid

Journal of Bacteriology ◽

10.1128/jb.186.12.3785-3793.2004 ◽

2004 ◽

Vol 186 (12) ◽

pp. 3785-3793 ◽

Cited By ~ 10

Author(s):

T. Betteridge ◽

J. Yang ◽

A. J. Pittard ◽

J. Praszkier

Keyword(s):

Escherichia Coli ◽

Binding Sites ◽

Origin Of Replication ◽

Initiator Protein ◽

Origin Of Dna Replication ◽

Replication Initiator Protein ◽

Replication Initiator

ABSTRACT The replication initiator protein RepA of the IncB plasmid pMU720 was shown to induce localized unwinding of its cognate origin of replication in vitro. DnaA, the initiator protein of Escherichia coli, was unable to induce localized unwinding of this origin of replication on its own but enhanced the opening generated by RepA. The opened region lies immediately downstream of the last of the three binding sites for RepA (RepA boxes) and covers one turn of DNA helix. A 6-mer sequence, 5′-TCTTAA-3′, which lies within the opened region, was essential for the localized unwinding of the origin in vitro and origin activity in vivo. In addition, efficient unwinding of the origin of replication of pMU720 in vitro required the native positioning of the binding sites for the initiator proteins. Interestingly, binding of RepA to RepA box 1, which is essential for origin activity, was not required for the localized opening of the origin in vitro.

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In Vitro Identification of Rns-Regulated Genes

Journal of Bacteriology ◽

10.1128/jb.184.4.1196-1199.2002 ◽

2002 ◽

Vol 184 (4) ◽

pp. 1196-1199 ◽

Cited By ~ 10

Author(s):

George P. Munson ◽

Lisa G. Holcomb ◽

Heather L. Alexander ◽

June R. Scott

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Binding Sites ◽

Genomic Dna ◽

Maltose Binding Protein ◽

Open Reading Frame ◽

Reading Frame ◽

Fusion Analysis

ABSTRACT To identify Rns-regulated genes, a maltose binding protein (MBP)-Rns fusion protein was used to isolate DNA fragments from enterotoxigenic Escherichia coli genomic DNA that carry Rns binding sites. In vivo transcription fusion analysis shows that Rns positively regulates the expression of the open reading frame of yiiS, which lies immediately downstream of one MBP-Rns-bound fragment.

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