Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor

The mutant cIts genes from seven different λcIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/ or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25°C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures

BlsA integrates light and temperature signals into iron metabolism through fur in the human pathogen Acinetobacter baumannii

Light modulates global features of the important human pathogen Acinetobacter baumannii lifestyle including metabolism, tolerance to antibiotics and virulence, most of which depend on the short BLUF-type photoreceptor BlsA. In this work, we show that the ability to circumvent iron deficiency is also modulated by light at moderate temperatures, and disclose the mechanism of signal transduction by showing that BlsA antagonizes the functioning of the ferric uptake regulator (Fur) in a temperature-dependent manner. In fact, we show that BlsA interacts with Fur in the dark at 23 °C, while the interaction is significantly weakened under blue light. Moreover, under iron deprived conditions, expression of Fur-regulated Acinetobactin siderophore genes is only induced in the dark in a BlsA-dependent manner. Finally, growth under iron deficiency is supported in the dark rather than under blue light at moderate temperatures through BlsA. The data is consistent with a model in which BlsA might sequester the repressor from the corresponding operator-promoters, allowing Acinetobactin gene expression. The photoregulation of iron metabolism is lost at higher temperatures such as 30 °C, consistent with fading of the BlsA-Fur interaction at this condition. Overall, we provide new understanding on the functioning of the widespread Fur regulator as well as short-BLUFs.Fil: Tuttobene, Marisel R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; ArgentinaFil: Cribb, Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mussi, María Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentin

The positive control of ilvC expression in E. coli K-12

The mechanism of ilvC expression in Escherichia coli was investigated. To carry out this work several different approaches were used. Firstly, sequencing of the ilvY2143 allele which carries a mutation that makes ilvC expression constitutive was completed. The location of the mutation was determined to be at the 5 end of the gene. It is a single base substitution (G to A) at position 87 (counted from the transcription startpoint of ilvY). This results in a change of the codon for one amino acid. Glutamine in wild-type ilvY protein is replaced by lysine in the constitutive one. This substitution in the polypeptide of the upsilon protein (product of the ilvY gene) was found to be solely responsible for making the up silon protein independent of the ilvC gene substrates (a-acetohydroxybutyrate or acetolactate) needed for ilvC induction. Two approaches were used to determine the direction of ilvY trancription. One of these employed a gene fusion technique which involves two DNA fragments of ilvY being fused separately to a promoterless lacZ gene, then monitoring the expression of lacZ. The other approach involved the labeling of the upsilon protein withes -methionine after expression of ilvY in a T7 RNA polymerase dependent promoter system. DNA-binding activity of upsilon protein was investigated. This was carried out in two assays, filter binding and gel retardation assays. These assays were employed to monitor purification of upsilon protein to near homogeneity. Upsilon protein has a subunit size of 35 kd and a native molecular weight of approximately 211 kd, suggesting upsilon exists as a hexamer. Finally, in vitro activities of the upsilon protein were tested using transcriptional and coupled transcription-translation assays. Upsilon protein was shown to cause elevation of ilvC transcription. Two models for the action of the upsilon protein in regulating the transcription of the ilvYC are proposed

Accurate Genetic Switch in Escherichia coli: Novel Mechanism of Regulation by Co-repressor

Understanding a biological module involves recognition of its structure and the dynamics of its principal components. In this report we present an analysis of the dynamics of the repression module within the regulation of the trp operon in Escherichia coli. We combine biochemical data for reaction rate constants for the trp repressor binding to trp operator and in vivo data of a number of tryptophan repressors (TrpRs) that bind to the operator. The model of repression presented in this report greatly differs from previous mathematical models. One, two or three TrpRs can bind to the operator and repress the transcription. Moreover, reaction rates for detachment of TrpRs from the operator strongly depend on tryptophan (Trp) concentration, since Trp can also bind to the repressor-operator complex and stabilize it. From the mathematical modeling and analysis of reaction rates and equilibrium constants emerges a high-quality, accurate and effective module of trp repression. This genetic switch responds accurately to fast consumption of Trp from the interior of a cell. It switches with minimal dispersion when the concentration of Trp drops below a thousand molecules per cell

Urate responsive MarR homologs

Differential gene expression in response to internal and external stimuli is studied in detail to understand the intricate mechanisms underlying response to various environmental stressors in microorganisms. MarR family transcriptional regulators have been studied for their involvement in such mechanisms. This work elucidates the mechanism of urate-induced attenuation of DNA binding of HucR, a MarR homolog, and extends this mechanism to describe a novel subfamily of MarR homologs responsive to urate, proposing a physiological relevance of utilizing urate as a signaling molecule. HucR (hypothetical urate regulator) binds to the shared promoter region between uricase and hucR genes. It has high specificity for urate in attenuation of DNA binding. The ligand-binding site in HucR was identified using molecular-dynamics guided mutational analysis, leading to a proposed mechanism for the attenuation of DNA binding upon interaction of urate. According to this model, urate is anchored in the binding pocket by W20 and R80 while a charge-repulsion displaces D73, which propagates the conformational change to the DNA recognition helix. A possible extension of this mechanism to other MarR homologs was examined through homology search where a number of MarR homologs were identified as conserving the residues involved in urate binding. Further, they show high sequence identity in helix-3, which includes the conserved aspartic acid residue and in the DNA recognition helix, a sequence conservation that correlates to the conservation of bases in their proposed 18 bp consensus dyadic-binding site. To further investigate this phenomenon, Agrobacterium tumefaciens-encoded PecS, which conserves these residues, was studied in detail. PecS binds to the shared promoter region between the genes pecS and pecM while urate attenuates DNA binding in vitro and elevates the transcript levels in vivo. This study thus identifies a novel subfamily of MarR family transcription factors that bind urate and proposes a novel signalling function of urate, wherein invading bacteria utilize urate produced by the host to promote successful host colonization

Formation of heterodimers between wild type and mutant trp aporepressor polypeptides of Escherichia coli

Availability of the three-dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that from the tryptophan binding pocket. Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid. Equilibrium dialysis with 14 C-tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants. The results obtained indicate that replacement of theronine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two-and four-fold, respectively. Replacement of ariginine 54 by histidine (RH84) or glycine 85 by ariginine (GR85) results in complete loss of tryptophan binding activity. Purified mutant and wild type aporepressors were used in vitro heterodimer studies. The trp repressor of E. coli functions as a stable dimer. A large number of trp repressor mutants prduces defective repressors that are transdominant to the wild type repressor in vivo. The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits. An in vitro assay was developed to detect and measure heterodimer formation. Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels. Aporepressors readily formed heterodimer formation upon treatment at 65°C for 3 minutes. Heterodimer formation was significantly retarded by the presence of the corepressor, L-tryptophan. Indole-3-propionic acid, 5-methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation. These results indicate that the presence of the indole moiety in the corepressor binding pocket increases the stability of the dimer.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38518/1/340040304_ftp.pd

The Structure of the Transcriptional Repressor KstR in Complex with CoA Thioester Cholesterol Metabolites Sheds Light on the Regulation of Cholesterol Catabolism in Mycobacterium tuberculosis

Cholesterol can be a major carbon source for Mycobacterium tuberculosis during infection, both at an early stage in the macrophage phagosome and later within the necrotic granuloma. KstR is a highly conserved TetR family transcriptional repressor that regulates a large set of genes responsible for cholesterol catabolism. Many genes in this regulon, including kstR, are either induced during infection or are essential for survival of M. tuberculosis in vivo. In this study, we identified two ligands for KstR, both of which are CoA thioester cholesterol metabolites with four intact steroid rings. A metabolite in which one of the rings was cleaved was not a ligand. We confirmed the ligand-protein interactions using intrinsic tryptophan fluorescence and showed that ligand binding strongly inhibited KstR-DNA binding using surface plasmon resonance (IC50 for ligand = 25 nM). Crystal structures of the ligand-free form of KstR show variability in the position of the DNA-binding domain. In contrast, structures of KstR·ligand complexes are highly similar to each other and demonstrate a position of the DNA-binding domain that is unfavorable for DNA binding. Comparison of ligand-bound and ligand-free structures identifies residues involved in ligand specificity and reveals a distinctive mechanism by which the ligand-induced conformational change mediates DNA release