Biomedical subjects

W Hillen

Publications and source records attributed to W Hillen.

At least 91 records · Page 5Linked to original sources

PubMed1995

Transcriptional activation by tetracyclines in mammalian cells.

A transcriptional transactivator was developed that fuses the VP16 activation domain with a mutant Tet repressor from Escherichia coli. This transactivator requires certain tetracycline (Tc) derivatives for specific DNA binding. Thus, addition of doxycycline to HeLa cells that constitutively synthesized the transactivator and that contained an appropriate, stably integrated reporter unit rapidly induced gene expression more than a thousandfold. The specificity of the Tet repressor-operator-effector interaction and the pharmacological characteristics of Tc's make this regulatory system well suited for the control of gene activities in vivo, such as in transgenic animals and possibly in gene therapy.

Amino Acid Sequence↗

PubMed1995

Extracellular expression of native human anti-lysozyme fragments in Staphylococcus carnosus.

Xylose-inducible vectors have been constructed for extracellular production of antibody fragments in Staphylococcus carnosus. The pre-pro sequence of S. hyicus lipase was taken as secretional signal sequence, and the S. xylosus Xyl repressor was used to confer xylose inducibility of transcription. Cleavage sites for the IgA protease were engineered between the pre-pro sequence and the antibody fragments to permit removal of the pro sequence. Extracellular expression of the light chain and the Fd fragment of a chimeric Fab fragment containing the variable regions of the anti-lysozyme antibody D1.3 was achieved with these vectors. The pro sequence could be removed from the expression product by IgA protease treatment. When the light chain and the Fd fragment were co-secreted as a protein fusion they accumulated in a structure capable of heterodimerization after IgA cleavage. This fusion contains the pre-pro sequence followed by the light chain, a second IgA site and the Fd fragment.

Antibodies↗

PubMed1995

In vitro binding of the CcpA protein of Bacillus megaterium to cis-acting catabolite responsive elements (CREs) of gram-positive bacteria.

Using DNA band migration retardation assays, specific binding of the CcpA protein of Bacillus megaterium to the cis-acting catabolite responsive element (CRE) of the xyl operon of B. subtilis has been demonstrated. Binding of CcpA was specifically inhibited by addition of unlabeled DNA fragments containing CREs of other operons but not by DNA fragments lacking a CRE. Binding was stimulated by high concentrations of phosphate, pyrophosphate, and organic phosphate esters and specifically inhibited by serine phosphorylated HPr and its conformational analogue, S46D HPr. This report therefore documents the specific binding of CcpA to a target CRE and defines its regulation by HPr(ser-P) and phosphorylated metabolites.

Bacillus megaterium↗

PubMed1995

The complex formed between Tet repressor and tetracycline-Mg2+ reveals mechanism of antibiotic resistance.

In recent years Gram-negative bacteria have developed several resistance mechanisms against the broad-spectrum antibiotic tetracycline (Tc). The most abundant mechanism involves a membrane-associated protein (TetA) that exports the antibiotic out of the bacterial cell before it can attach to the ribosomes and inhibit polypeptide elongation. The expression of the TetA protein is regulated by the Tet repressor (TetR). It occurs as a homodimer and binds with two alpha-helix-turn-alpha-helix motifs (HTH) to two tandemly orientated DNA operators, thereby blocking the expression of the associated genes, one encoding for TetA and the other for TetR. If Tc in complex with a divalent cation binds to TetR, a conformational change occurs and the induced TetR is then unable to bind to DNA. TetR of class D, TEtRD, was cocrystallized with tetracycline (7HTc) and Mg2+ in space group I4(1)22 and studied by X-ray diffraction. One TetRD monomer occupies the crystal asymmetric unit, and the dimer is formed by a crystallographic 2-fold rotation. The crystal structure was determined by multiple isomorphous replacement at 2.5 A resolution, and on this basis the structure of the nearly isomorphous complex with 7-chlorotetracycline, TetRD/(Mg 7CITc)+, has been refined to an R-factor of 18.3% using all reflections to 2.1 A resolution. TetRD folds into ten alpha-helices with connecting turns and loops. The N-terminal three alpha-helices of the repressor form the DNA-binding domain, including the HTH with an inverse orientation compared with HTH in other DNA-binding proteins. The distance of 39 A between the two recognition helices explains the inability of the induced TetR to bind to B-form DNA. The core of the protein is formed by helices alpha 5 to alpha 10. It is responsible for dimerization and contains, for each monomer, a binding pocket that accommodates Tc in the presence of a divalent cation. The structure of the TetRD/(Mg 7CITc)+ complex reveals the octahedral coordination of Mg2+ by Tc (chelating O-11, and O-12), His100 N epsilon and by three water molecules; in addition there is an extended network of hydrogen bonding and van der Waals interactions formed between 7CITc and TetR. The detailed view of the Tc-binding pocket and the interactions between the antibiotic and the repressor offers the first solid basis for rational tetracycline design, with the aim of circumventing resistance.

Amino Acid Sequence↗

PubMed1995

Proximity probing of Tet repressor to tet operator by dimethylsulfate reveals protected and accessible functions for each recognized base-pair in the major groove.

We have tracked the path of Tet repressor across the major groove in the complex with tet operator. This was done by a methylation protection analysis of nine tet operator mutants containing replacements by a G residue of each nucleotide in base-pairs important for Tet repressor recognition. We demonstrated sequence-specific binding of Tet repressor to these operator mutants using DNA retardation assays and the protection of the wild-type +2G residue from methylation. Hydroxyl radical cleavage protection analysis of the Tet repressor-tet operator complexes indicated identical, or at least very similar, locations of the DNA reading head across the major groove of wild-type and mutant operator DNA. Methylation protection occurred at the G residues in positions +3, +4, -5 and -6, whereas the G residues in the respective opposite strands showed enhanced methylation. These results show that most amino acid side-chains of Tet repressor are in close proximity to only one base of each base-pair in the major groove of tet operator. The Tet repressor mutant PS39 gave a changed methylation protection pattern at base-pair four of tet operator indicating that the residue at this position can contact either base at this base-pair depending on the amino acid side-chain present. Tet repressor mutants QA38 and TA40 with a loss of specificity phenotype gave the same methylation protection profile as wild-type TetR confirming that this experiment scores proximity rather than chemical interaction. The excellent agreement of these results with those obtained in genetic analyses demonstrates that this method yields a high-resolution proximity pattern of Tet repressor with tet operator and that it may be generally applicable for the analysis of protein-DNA complexes.

Base Composition↗

PubMed1995

Proximity mapping of the Tet repressor-tetracycline-Fe2+ complex by hydrogen peroxide mediated protein cleavage.

We demonstrate in a quantitative in vitro induction assay that tetracycline-Fe2+ is a more than 1000-fold stronger inducer of Tet repressor compared to tetracycline-Mg2+. Oxidative cleavage of the Tet repressor-tetracycline-Fe2+ complex with H2O2 and ascorbate results in an Fe(2+)-dependent specific fragmentation of the protein. The maximal yield of about 15% and a reaction time of less than 30 s are only observed in the presence of the drug, whereas about 1% cleavage is obtained after 30 min in the presence of Fe2+ without tetracycline. Cleavage is not inhibited by several radical scavengers, suggesting a highly localized reactivity of the redox-active oxo intermediates in the proximity of the Fe(2+)-tc chelater where they are generated. The products can be separated by HPLC only after denaturation, indicating that the complex is not disrupted by cleavage. Residues at which the cleavage takes place are identified using the masses of the fragments determined by electrospray mass spectrometry and their N-terminal sequences. The major cleavage site maps to residues 104 and 105 of Tet repressor. Less efficient cleavages occur at residues 56 and 136, and the least efficiently cleaved sites are around residues 144 and 147. The cleavage efficiencies correlate to the distances and orientations of the respective peptide bonds to Mg2+ in the crystal structure of the Tet repressor-tetracycline-Mg2+ complex. We discuss potential reaction mechanisms leading to protein cleavage.

Amino Acid Sequence↗

PubMed1995

Characterization of non-inducible Tet repressor mutants suggests conformational changes necessary for induction.

Non-inducible tetracycline repressor (TetR) mutants were grouped in three structurally distinct classes. We quantitated in vivo operator binding, inducibility, and in vitro tetracycline binding of mutants from each class. Mutation of residues close to tetracycline (class 1) leads to reduced affinity for the drug. Mutation of residues located at the connection of the DNA-reading head with the protein core (class 2) and at the dimerization interface (class 3) bind inducer with the same affinity as wild-type TetR. These mutations interfere with the induced, but not the operator-binding conformation of TetR. The affinity of some class 1 mutants for tetracycline is less affected than their inducibility, suggesting that the mutated residues are important for triggering those conformational changes necessary for induction.

Amino Acid Sequence↗

PubMed1995

Genetic organization, nucleotide sequence and regulation of expression of genes encoding phenol hydroxylase and catechol 1,2-dioxygenase in Acinetobacter calcoaceticus NCIB8250.

We have mutated Acinetobacter calcoaceticus NCIB-8250 to growth deficiency on phenol as sole carbon source and isolated genes with similarity to phenol hydroxylase and catechol 1,2-dioxygenase by complementation. Sequence analysis reveals the presence of six open reading frames (ORFs) with similarities to a Pseudomonas multicomponent phenol hydroxylase which are followed by an ORF with similarity to catA from A. calcoaceticus ADP1. Transformation of these genes to ADP1 confers the ability to grow at the expense of phenol as sole carbon source. Primer extension analysis indicates phenol-inducible transcription from an RpoN-dependent promoter sharing sequence similarity with the sigma 54 consensus promoter sequence, except that the -12 box is GG instead of GC. A catA::lacZ transcriptional fusion shows the same induction profile for beta-galactosidase expression as transcription from the sigma 54-dependent promoter. This result suggests that catA is cotranscribed in the same operon with the phenol hydroxylase-encoding genes and is consistent with the fact that no apparent additional promoter is found for catA by sequence analysis or primer extension. Catechol 1,2-dioxygenase activity is induced in NCIB8250 by benzoate, whereas beta-galactosidase expression from the catA::lacZ fusion is not. This observation leads to the hypothesis that two differentially regulated catA genes should be present in that strain.

Acinetobacter calcoaceticus↗

PubMed1995

Deletion mutagenesis of Tn 10 Tet repressor--localization of regions important for dimerization and inducibility in vivo.

The gene for the Tn 10 Tet repressor (TetR) was subjected to deletion mutagenesis. Screening for a transdominant operator-binding negative phenotype yielded 10 mutants with internal deletions. Three deletions extend from residue D5 to residues L41, W75, or Q76, respectively, and two contain deletions of the alpha-helix-turn-alpha-helix DNA-binding motif. Five deletions range from residue K84 to residues between R87 and K98. Since residues from the N-terminus up to position 98 are not necessary for dimerization, this must take place in the C-terminal half of the protein. Ability to dimerize was probed by introducing ochre nonsense codons (oc) at residues G138, H151, E159, I174, or K202. Koc202 shows wild-type in vivo operator-binding and inducibility by tetracycline indicating that the six C-terminal residues of TetR are not important for activity. Mutants with longer C-terminal truncations are inactive and not transdominant. They show reduced steady-state protein levels and are probably impaired in folding and degraded in vivo. Two mutants (delta151-166, delta164-166) with deletions in a region variable in primary structure and length among Tet repressors from different resistance determinants bind tet operator efficiently, but are not inducible by tetracycline. This result indicates that these residues are not important for dimer formation in the operator-binding form.

Amino Acid Sequence↗

PubMed1995

Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria?

Three components involved in catabolite repression (CR) of gene expression in Bacillus have been identified. The cis-acting catabolite responsive element (CRE), which is present in many genes encoding carbon catabolic enzymes in various species of the Gram-positive bacteria, mediates CR of several genes in Bacillus subtilis, Bacillus megaterium, and Staphylococcus xylosus. CR of most genes regulated via CRE is also affected by the trans-acting factors CcpA and HPr. Similarities between CcpA and Lac and Gal repressors suggest binding of CcpA to CRE. HPr, a component of the phosphoenolpyruvate:sugar phosphotransferase system, undergoes regulatory phosphorylation at a serine residue by a fructose-1,6-diphosphate-activated kinase. A mutant of HPr, which is not phosphorylatable at this position because of an exchange of serine to alanine, lacks CR of several catabolic activities. This mutant phenotype is similar to the one exhibited by a ccpA mutant. Direct protein-protein interaction between CcpA and HPr(Ser-P) was recently demonstrated and constitutes a link between metabolic activity and CR.

Amino Acid Sequence↗

PubMed1995

Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in gram-positive bacteria.

CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram-positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA-his immobilized on a Ni-NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phospho-carrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His-15 in a PEP-dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser-46 in an ATP-dependent reaction catalysed by a metabolite-activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His-15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl-phosphorylated HPr required the presence of fructose 1,6-bisphosphate. These findings suggest that carbon catabolite repression in Gram-positive bacteria is a protein kinase-triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P-ser-HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His-15 also suggests a link between carbon catabolite repression and PTS transport activity.

Amino Acid Sequence↗

PubMed1995

Cloning, expression and functional analyses of the catabolite control protein CcpA from Bacillus megaterium.

A mutant of Bacillus megaterium relieved from catabolite repression has been used to clone ccpA from B. megaterium by complementation. ccpA is the first gene of a presumed operon, in which it is followed by the motA homologue ORF1 and the motB homologue ORF2. The mutation maps in the 3'-terminal region of ccpA, where an in-frame duplication of 84 nucleotides located between two 9 bp direct repeats leads to an insertion of 28 amino acids near the C-terminus of CcpA. An in-frame deletion of 501 bp in ccpA exhibits the same phenotype as the 84 bp duplication. Deletion of ORF1 and ORF2 does not yield an apparent phenotype. A single-copy ccpA::lacZ transcriptional fusion is constitutively expressed, independent of whether the growth medium triggers catabolite repression or not. The ccpA mutation leads to relief of catabolite repression exerted by glucose, fructose, mannitol, glucitol and glycerol, whereas only smaller effects were found with ribose, citrate and glutamate. The respective growth rates on these carbon sources are uniformly reduced to a generation time of about 90 min in the ccpA mutant. Catabolite repression of a plasmid-encoded xylA::ccpA fusion is less efficient than that of a xylA::lacZ fusion in the same vector. Furthermore, overproduction of CcpA decreases catabolite repression of a single-copy xylA::lacZ fusion approximately twofold. Thus, overexpression of CcpA may be counterproductive for catabolite repression, supporting the hypothesis that CcpA by itself may not bind sufficiently strongly to the cis-active catabolite-responsive element to exert catabolite repression.

Bacillus megaterium↗

PubMed1995

Glucose and glucose-6-phosphate interaction with Xyl repressor proteins from Bacillus spp. may contribute to regulation of xylose utilization.

The xyl operons of several gram-positive bacteria are regulated at the level of transcription by xylose-responsive repressor proteins (XylR). In addition, they are catabolite repressed. Here, we describe a mechanism by which glucose metabolism can affect both regulatory mechanisms. Glucose-6-phosphate appeared to be an anti-inducer of xyl operon transcription, since it could compete with xylose in interaction in vitro with XylR from Bacillus subtilis, B. megaterium, and B. licheniformis. On the other hand, glucose was a low-efficiency inactivator of XylR from B. subtilis and B. megaterium and a weak anti-inducer of XylR from B. licheniformis. Thus, the chemical nature of the substituent at C-5 of xylose and the primary structure of XylR determine the effect of these compounds on xyl operon transcription.

Bacillus↗

PubMed1995

Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus.

The tetracycline repressor (TetR)-regulated gene expression system from Escherichia coli was used to control gene expression in recombinant human cytomegalovirus (HCMV). To adapt the TetR system in HCMV, derivatives of the viral US11 (early) gene promoter, which controls the beta-glucuronidase reporter gene, were constructed by systematic insertion of the tetracycline operator (tetO) sequences. Gene expression from constructs containing two or three appropriately placed tetO sequences adjacent to the TATA box were efficiently repressed by a TetR-VP16 fusion protein (tTA) in a transient expression system. Efficient repression (50- to 120-fold) also occurred in tTA-expressing stably transfected human cells which were infected with recombinant HCMV containing a US11 promoter surrounded by three tetO sequences. The tTA-mediated gene repression was relieved in the presence of 1 microgram of tetracycline per ml. The results of this study are significant in three respects. (i) This is the first demonstration that a TetR-derived protein can be used to efficiently repress gene expression in a mammalian system. (ii) Efficient repression was dependent on the presence of the transcriptional activation domain from the herpes simplex virus type 1 VP16 protein. (iii) The ability to regulate gene expression in a controlled fashion in order to elucidate viral gene function is an important development in the HCMV field. The tTA-mediated gene repression system may be extremely useful for creating host-range mutants in essential genes in order to study their role in the HCMV replicative cycle, a system that is otherwise exceedingly difficult to genetically dissect.

Base Sequence↗

PubMed1994

Transcription of the xyl operon is controlled in Bacillus subtilis by tandem overlapping operators spaced by four base-pairs.

The expression of xylose utilization in Bacillus subtilis is regulated at the level of transcription by xylose dependent Xyl repressor-xyl operator interaction. We have structurally and functionally characterized the binding sites of Xyl repressor in the xyl regulatory region. Methylation and hydroxyl radical protection and ethylation interference of binding suggests tandem overlapping xyl operators spaced by four base-pairs. A mutational inactivation of each and both operators was performed. DNA retardation experiments with these mutants confirmed the existance of two binding sites. They can be simultaneously occupied, despite their overlapping, intertwined organization. In vivo repressor titration and regulation of indicator gene expression by the xylO mutants confirmed that both binding sites contribute to regulation of the xyl operon. The protection and interference patterns of both sites are identical and indicate binding of a repressor oligomer to one side of B-form DNA of each operator. A tandem overlapping arrangement of two operators is also found in the xyl regulatory sequences of Bacillus megaterium, Staphylococcus xylosus and Lactobacillus pentosus. The xyl operon of Bacillus licheniformis contains a similar element in which the second operator is more diverged. This high degree of conservation among bacteria of different genera supports the conclusion that a tandem overlapping arrangement of xyl operators contributes to efficient regulation.

Amino Acid Sequence↗

PubMed1994

Sequences of ccpA and two downstream Bacillus megaterium genes with homology to the motAB operon from Bacillus subtilis.

A regulatory gene with 69% nucleotide sequence identity to the Bacillus subtilis ccpA was cloned from Bacillus megaterium by complementation of a mutant relieved of catabolite repression. Sequencing of the gene and its adjacent regions revealed two additional open reading frames (ORFs) downstream from ccpA. These three genes are presumably in one operon. ORF1 and ORF2 show homology to two genes downstream from ccpA in B. subtilis, as well as to the B. subtilis motA and motB genes, respectively.

Amino Acid Sequence↗

PubMed1994

Oligo[d(C).(G)] runs exhibit a helical repeat of 11.1 bp in solution and cause slight DNA curvature when properly phased.

We have inserted d(C)10 in a set of DNA fragments with bent segments on both ends, which are rotated with respect to each other by base pair wise increasing insertions. The electrophoretic mobilities on polyacrylamide gels of these DNA fragments were used to identify insertion sizes with cis conformations of the bent ends. These experiments revealed a helical repeat in solution of d(C).d(G) tracts of 11.1 +/- 0.08 bp. The electrophoretic mobilities of ligation ladders with properly phased d(C)5 and d(C)16 runs demonstrate a small but clearly detectable curvature of these fragments.

Base Sequence↗

PubMed1994

Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance.

The most frequently occurring resistance of Gram-negative bacteria against tetracyclines is triggered by drug recognition of the Tet repressor. This causes dissociation of the repressor-operator DNA complex and enables expression of the resistance protein TetA, which is responsible for active efflux of tetracycline. The 2.5 angstrom resolution crystal structure of the homodimeric Tet repressor complexed with tetracycline-magnesium reveals detailed drug recognition. The orientation of the operator-binding helix-turn-helix motifs of the repressor is inverted in comparison with other DNA binding proteins. The repressor-drug complex is unable to interact with DNA because the separation of the DNA binding motifs is 5 angstroms wider than usually observed.

Antiporters↗