bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of systematic variation of the minimal Escherichia coli met consensus operator site: in vivo and in vitro met repressor binding
Authors: Wild Catherine M. , McNally Teresa , Phillips Simon E. V. , Stockley Peter G. 
Published: November 2003

Summary We have produced a set of sequence variants based upon the idealized, minimal Escherichia coli met operator in which each position within the basic recognition unit, the 8bp met box (dAGACGTCT), has been changed to all other possible sequences containing single symmetrical base substitutions. The effects of these sequence variations have been assayed in vivo by monitoring the production of β‐galactosidase from a standard promoter regulated by the operator variants, and in vitro by gel‐retardation assay. The two sets of data are consistent and correlate well with expectations based on the three‐dimensional structure of the holorepressor bound to a minimal idealized operator and the results of in vitro evolution experiments. Comparison with two natural operators, metA and metC, suggests that in vivo, with non‐consensus operators, the repressor binds to at least four consecutive met boxes.
A cyclophilin‐like peptidyl‐prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression
Authors: Schmidt Bettina , Tradler Thomas , Rahfeld Jens‐U. , Ludwig Birgit , Jain Bunty , Mann Karlheinz , Rücknagel K. Peter , Janowski Bernhard , Schierhorn Angelika , Küllertz Gerhard , Hacker Jörg , Fischer Gunter 
Published: November 2003

Summary Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506‐binding protein, FKBP25mem), belongs to the enzyme family of peptidyl‐prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N‐terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164‐amino‐acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19‐amino‐acid polypeptide extension including a 6‐histi‐dine tag and an enterokinase cleavage site exhibits
Flagellar assembly in Salmonella typhimurium
Authors: Aizawa Shin‐Ichi 
Published: November 2003

SummaryThe bacterial flagellum is a motility apparatus in which a long helical filament ‐ the propeller ‐ is driven by a rotary motor embedded in the cell surface. Out of more than 40 genes required for construction of a fully functional flagellum in Salmonella typhimurium, only 18 gene products have been identified in the mature structure. Some other flagellar proteins play logistical roles during construction, which involves the selective export of flagellar components through a central hole in the flagellum. The whole structure is constructed from base to tip by linear assembly; that is, by adding new components on the growing end, resulting in the distal growth of each substructure. Components of the substructures do not necessarily self‐assemble, but often demand the help of other proteins. Recent progress in the understanding of flagellar assembly, which has been most extensively studied in S. typhimurium, is reviewed.
Expression of the groESL operon is cell‐cycle controlled in Caulobacter crescentus
Authors: Avedissian Marcelo , Gomes Suely Lopes 
Published: November 2003

SummaryThe Caulobacter crescentus groESL operon was cloned, sequenced and found to be homologous to previously described groES and groEL genes and proteins. The size of the groESL‐specific transcript (2.3 kb) suggested that groES and groEL of C. crescentus are organized in a bicistronic operon. Heat‐shock induction of groESL mRNA is not transient, high levels of the transcript can be observed after 2 h at 40°C. Prlmer extension experiments showed that transcription initiated at two sites. Only the start site closer to the groES coding region was highly induced during heat shock. The promoter corresponding to the heat‐shock‐inducible transcript has −10 and −35 regions very similar to Escherichia coliσ32 promoters. At normal temperatures, transcription of the groESL operon is cell‐cycle controlled and both transcripts increase co‐ordinately in pre‐divisional cells. Transcription fusions with a lacZ reporter gene and deletions within the promoter region of the groESL operon have shown that no sequences upstream of the heat‐shock promoter are necessary for temporal control. An 11 bp inverted repeat, located between the heat‐shock promoter and the translation start site of groES and very similar to inverted repeats found in front of several heat‐shock genes of other bacteria, may play a role in cell‐cycle control of C. crescentus groESL expression.
FNR‐DNA interactions at natural and semi‐synthetic promoters
Authors: Green J. , Irvine A. S. , Meng W. , Guest J. R. 
Published: November 2003

SummaryTwo rapid and convenient methods have been developed for the amplification and purification of FNR, the anaerobic transcription regulator of Escherichia coli The overproduced proteins resemble wild‐type FNR in their basic properties: oligomeric state, iron contents (up to 2.7 atoms per monomer), DNA‐binding affinities and ability to activate transcription. However, unlike previous preparations, FNR could be isolated in a form containing up to 0.25 atoms of acid‐labile sulphur per monomer. Incorporation of iron increased the Mr of FNR from 28 000 to 40 000. Under anaerobic conditions, reconstituted FNR exhibited absorption maxima at 315nm and 420 nm, which were replaced by a broad absorbance from 380 to 440 nm under aerobic conditions. These observations indicate that FNR contains one redox‐sensitive [3Fe 4S] or [4Fe 4S] centre per monomer. Footprints of FNR‐dependent promoters (ansB, fdn, fnr, narG, pflP6, pflP7 and nirB) showed protection at all of the predicted FNR sites except the pflP7 (‐57.5), ansB (‐74.5) and nirB (‐89.5) sites. An unpredicted second binding site was detected at ‐57.5 in the narG promoter. Hypersensitive sites within regions of FNR protection indicated that FNR bends DNA in a similar way to CRP. Promoters containing binding sites for FNR (FF), CRP (CC) or hybrid sites (CF or FC) were footprinted with FNR and two derivatives (FNR‐610 and FNR‐573) which activate the CCmeIR promoter in vivo. FNR preferentially protected the FNR site (FF) whereas FNR‐610 preferred CC and FNR‐573 interacted with equal affinity at all sites.
In vitro selection of optimal AbrB‐binding sites: comparison to known in vivo sites indicates flexibility in AbrB binding and recognition of three‐dimensional DNA structures
Authors: Xu Ke , Strauch Mark A. 
Published: November 2003

SummaryThe AbrB protein of Bacillus subtilis regulates expression of numerous genes, primarily through specific binding interactions to DNA regions containing transcriptional promoters. Although over 15 target regions for AbrB binding to chromosomally located sequences have been analysed by DNase I footprinting, no obvious consensus sequence or motif has yet emerged from their examination. Using in vitro selection techniques, we have isolated optimal AbrB‐binding sites from oligonucleotides containing 22 or 44 random base pairs. The best of these sites have an apparent in vitro Kd which is fivefold lower than a similar‐sized DNA fragment containing the sequence corresponding to the AbrB‐binding site on the spoOE gene. We tested one of the sites in vivo and found that it confers AbrB‐mediated control upon a promoter not normally regulated by AbrB. In each of four separate trials, the selected sites possess motifs that converge to a simple consensus. It is argued that the nature and spacing of these motifs produce a type of three‐dimensional DNA structure recognizable by AbrB, and that known in vivo sites, which lack these motifs, possess an approximation of the optimal structural determinant.
Purification of the Tn 10‐specified tetracycline efflux antiporter TetA in a native state as a polyhistidine fusion protein
Authors: Aldema Mila L. , McMurry Laura M. , Walmsley Adrian R. , Levy Stuart B. 
Published: November 2003

SummaryThe bacterial tetracycline‐resistance determinant from Tn 10 encodes a 43 kDa membrane protein, TetA, responsible for active efflux of tetracyclines. The tetA gene was cloned behind a T7 promoter/ac operator in a plasmid that provided fusion of TetA to a polyhis‐tidine‐carboxy terminal tail. A second plasmid provided a regulated T7 RNA polymerase. The specific activity of the TetA fusion protein was between 10–40% that of the wild‐type protein as assayed by tetracycline resistance in cells and by transport in membrane vesicles. The fusion protein, overproduced approximately 3–13‐fold, was purified by nickel chelation chromatography. Calculations from circular dichroism spectra of the purified protein solubilized in dodecylmaltoside gave an α‐helix content of 54–64%, close to the 68% predicted from the amino acid sequence by hydropathy analysis (12 membrane‐spanning helices) for the native protein in the membrane bilayer. Fluorescence studies showed binding activity of the purified protein to its substrate, the tetracycline analogue 13‐(cyclopentylthio)‐5‐hydroxy‐6‐α‐deoxyte‐tracycline. These findings suggested that the purified protein was in a native state.
Characterization of the CheAS/CheZ complex: a specific interaction resulting in enhanced dephosphorylating activity on CheY‐phosphate
Authors: Wang Hui , Matsumura Philip 
Published: November 2003

The cheA gene encodes two overlapping polypeptides with a common carboxyl terminus: CheAL and CheAS. CheAL plays a central role in the Escherichiacoli chemotaxis signalling pathway by autophosphorylation and transferring the phosphate to both CheY and CheB. On the other hand, the physiological functions of CheAS remain unknown.We have observed that overproduction of CheAS in wild‐type cells increased counterclockwise‐biased flagellar rotation, and this effect is dependent on the presence of CheZ. CheZ specifically facilitates CheY‐phosphate (CheY‐P) dephosphorylation and generates a smooth swimming signal. A physical interaction was detected between CheZ and CheAS in wild‐type cell lysates by immunoprecipitation. The CheAS/CheZ interaction does not require other chemotaxis components, as we could form the complex using purified CheAS and CheZ proteins. The ability of CheAS to bind to CheZ depends on its being in the reduced state. We found that under non‐reducing conditions, CheAS appears to form intermolecular disulphide bonds and loses the ability to bind to CheZ. Finally, the CheAS/CheZ complex formed in vitro shows a greater dephosphorylating activity on CheY‐P than does free CheZ.
Role of the conserved carboxy‐terminal α‐helix of Rad6p in ubiquitination and DNA repair
Authors: Dor Yuval , Raboy Bilha , Kulka Richard G. 
Published: November 2003

Summary
RAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin‐conjugating enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p require the ubiquitination of target proteins, but its substrates as well as other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C‐terminal α‐helix, which are conserved from yeast to man but are absent from most other yeast ubiquitin‐conjugating enzymes (Ubcs). This specific conservation suggests that the C‐terminal a‐helix is important for the unique activities of the Rad6p family of Ubcs. We have investigated the effects of mutating this highly conserved region on the ubiquitination of model substrates in vitro and on error‐free DNA repair in vivo. C‐terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the possibility that Rad6p interacts with its substrates in vivo by similar mechanisms. The distal part of the C‐terminal u‐helix is also essential for error‐free DNA repair in vivo. Overexpression of Rad18p, a single‐stranded DNA‐binding protein that also interacts with Rad6p, alleviates the DNA repair defects of the C‐terminal α‐helix mutants to different degrees. This indicates that the C‐terminal α‐helix of Rad6p mediates its interaction with Rad18p, an essential step in DNA repair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants affecting several functions of the 26S proteasome retain wild‐type capacity for error‐free DNA repair. This raises the possibility that ubiquitination by Rad6p in DNA repair does not target proteins for proteasomal degradation.
Role of stoichiometry between mRNA, translation factor SeIB and selenocysteyl‐tRNA in selenoprotein synthesis
Authors: Tormay P. , Sawers A. , Böck A. 
Published: November 2003

SummaryThe specialized translation factor SeIB forms a quaternary complex in vitro with selenocysteyl‐tRNASec, the selenoprotein mRNA and guanine nucleotides. To gain information on whether this complex is required for selenocysteine insertion in vivo we have studied the effect of unbalanced ratios of the individual components of the complex on UGA readthrough. It was found that overproduction of SeIB in an otherwise wild‐type genetic background reduced UGA read‐through to less than 1 %. Concomitant overexpression of seIC (the gene for selenocysteine‐specific tRNASec) completely reversed the inhibition. Truncation of SeIB from the C‐terminal end abolished function as a translation factor but the truncated molecules, when overproduced, were still able to suppress UGA read‐through. The inhibition was also reversed by overproduction of tRNASec. The most plausible explanation is that overproduction of SeIB impairs the statistics of formation of the quaternary complex and that the C‐terminally truncated molecules are still able to bind selenocysteyl‐tRNASec and remove it from the pool. The mRNA‐binding capacity, therefore, is physically separated from the selenocysteyl‐tRNA‐binding domain.
Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite‐responsive elements
Authors: Miwa. Yasuhiko , Nagura Kazuya , Eguchi Susumu , Fukuda Hirokazu , Deutscher Josef , Fujita Yasutaro 
Published: November 2003

SummaryCatabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis‐acting catabolite‐responsive element (cre) by a complex of CcpA, which is a member of the GalR‐LacI family of bacterial regulatory proteins, and the seryl‐phos‐phorylated form of HPr (P‐ser‐HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the ere in the first gene (gntR) of the gnt operon (credown), this operon contains another ere located in the promoter region (creup). A translational gntR‐lacZ fusion expressed under the control of various combinations of wild‐type and mutant credown and creup was integrated into the chromosomal amyE locus, and then catabolite repression of p‐galac‐tosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by creup was probably independent of catabolite repression exerted by credown; both creup and credown catabolite repression involved CcpA. Catabolite repression exerted by creup was independent of P‐ser‐HPr, and catabolite repression exerted by credown was partially independent of P‐ser‐HPr. DNase I footprinting experiments indicated that a complex of CcpA and P‐ser‐HPr did not recognize creup, in contrast to its specific recognition of credown. However, CcpA complexed with glucose‐6‐phosphate specifically recognized creup as well as credown, but the physiological significance of this complexing is unknown.
Molecular characterization of the Bacillus anthracis main S‐layer component: evidence that it is the major cell‐associated antigen
Authors: Mesnage Stéphane , Tosi‐Couture Evelyne , Mock Michèle , Gounon Pierre , Fouet Agnès 
Published: November 2003

Summary Bacillus anthracis, the aetiological agent of anthrax, is a Gram‐positive spore‐forming bacterium. The cell wall of vegetative cells of B. anthracis is surrounded by an S‐layer. An array remained when sap, a gene described as encoding an S‐layer component, was deleted. The remaining S‐layer component, termed EA1, is chromosomally encoded. The gene encoding EA1 (eag) was obtained on two overlapping fragments in Escherichia coli and shown to be contiguous to the sap gene. The EA1 amino acid sequence, deduced from the eag nucleotide sequence, shows classical S‐layer protein features (no cysteine, only 0.1% methionine, 10% lysine, and a weakly acidic pi). Similar to Sap and other Gram‐positive surface proteins, EA1 has three 'S‐layer‐homology’motifs immediately downstream from a signal peptide. Single‐ and double‐disrupted mutants were constructed. EA1 and Sap were co‐localized at the cell surface of the wild‐type bacilli. However, EA1 was more tightly bound than Sap to the bacteria. Electron microscopy studies and in vivo experiments with the constructed mutants showed that EA1 constitutes the main lattice of the B. anthracis S‐layer, and is the major cell‐associated antigen.
A two‐gene ABC‐type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore
Authors: Cheng Jianbo , Guffanti Arthur A. , Krulwich Terry Ann 
Published: November 2003

SummaryA transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na at elevated pH, was deficient in energy‐dependent Na extrusion. The capacity of the mutant JC901 for Na ‐dependent pH homeostasis was unaffected relative to the wild‐type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3 ‐terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane‐spanning regions at its C‐terminus, and exhibits modest sequence similarity to regions of eukaryotic Na+/H+ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP‐binding proteins that are components of transport systems of the ATP‐binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natAB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p‐chlorophenylhydrazone (CCCP), and, more modestly, by Na+, and K+, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na+‐sensitive phe‐notype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol‐resistance does not result from NatAB‐dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na+
DnaA protein stimulates polA gene expression in Escherichia coli
Authors: Quiñones Ariel , Wandt Gudrun , Kleinstäuber Sabine , Messer Walter 
Published: November 2003

SummaryThe polA gene of Escherichia coli encodes DNA polymerase I that is involved in DNA replication and repair. Despite the wide knowledge about structure and function of DNA polymerase I, there is little insight into the regulatory mechanisms involved in polA expression. DnaA is the initiator protein for DNA replication in E. coli. There are two putative DnaA‐binding sites within the extended promoter region of polA. In this work we studied the influence of altered levels of DnaA protein on polA expression. We found that DnaA overproduction increases polA expression in stationary‐phase cultures. The stimulation effect was independent of rpoS, which encodes the sigma factor for stationary‐phase‐inducible genes. However, it was modulated by ppGpp. Comparative S1 analyses revealed that the induction was based on transcriptional stimulation. Footprint‐ing experiments demonstrated that DnaA binds only to the proximal DnaA box near the polA promoter. These results suggest an additional role for DnaA as transcriptional activator of polA at least under certain physiological conditions.
Random shuttle mutagenesis: gonococcal mutants deficient in pilin antigenic variation
Authors: WIehr Ian J. , Seifert H. Steven 
Published: November 2003

SummaryShuttle mutagenesis has been adapted to randomly mutate the genome of Neisseria gonorrhoeae (gono‐coccus; Gc). A size‐restricted plasmid library of Gc strain FA1090 was mutated with the mini‐transposon mTnEGNS. Randomness was tested by checking for transposon insertion bias between vector and insert DNA, Gc transformation efficiency of individual mutated clones, and representation of unique clones before and after Gc transformation with a mutated pool of DNA. Mutants created by random shuttle mutagenesis were screened, using a colony‐based polymerase chain reaction assay, for the ability to undergo pilin antigenic variation. Out of 8064 mutants screened, 22 unique transposon insertion mutants were found to be antigenic variation deficient (Avd). The Avd mutants were separated into five types according to recombination defect‐associated phenotypes, including colony growth, natural DNA transformation competence, and repair of DNA damage caused by ultraviolet radiation.
Maltose‐binding protein interacts simultaneously and asymmetrically with both subunits of the Tar chemoreceptor
Authors: Gardina Paul J. , Bormans Arjan F. , Hawkins Murphy A. , Meeker Joshua W. , Manson Michael D. 
Published: November 2003

SummaryThe Tar chemotactic signal transducer of Escherichia coli mediates attractant responses to L‐aspartate and to maltose. Aspartate binds across the subunit interface of the periplasmic receptor domain of a Tar homodimer. Maltose, in contrast, first binds to the periplasmic maltose‐binding protein (MBP), which in its ligand‐stabilized closed form then interacts with Tar. Intragenic complementation was used to determine the MBP‐binding site on the Tar dimer. Mutations causing certain substitutions at residues Tyr‐143, Asn‐145, Gly‐147, Tyr‐149, and Phe‐150 of Tar lead to severe defects in maltose chemotaxis, as do certain mutations affecting residues Arg‐73, Met‐76, Asp‐77, and Ser‐83. These two sets of mutations defined two complementation groups when the defective proteins were co‐expressed at equal levels from compatible plasmids. We conclude that MBP contacts both subunits of the Tar dimer simultaneously and asymmetrically. Mutations affecting Met‐75 could not be complemented, suggesting that this residue is important for association of MBP with each subunit of the Tar dimer. When the residues involved in interaction with MBP were mapped onto the crystal structure of the Tar periplasmic domain, they localized to a groove at the membrane‐distal apex of the domain and also extended onto one shoulder of the apical region.