bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reverse transcriptase in bacteria
Authors: Lim D. , Maas W. K. 
Published: October 2006

SummaryReverse transcriptase, discovered in 1970 in retroviruses, has until recently been found only in eukaryotic organisms. Recently it was shown to occur in two groups of bacteria: myxobacteria and Escherichia coli. The gene for reverse transcriptase is part of a chromosomal genetic element that codes for the production of a branched DNA‐RNA compound. In this compound a single‐stranded DNA is connected to RNA at a specific G residue by a 2′‐5’phosphodiester linkage. The precursor for the DNA‐RNA compound is a folded messenger RNA, in which the specific G residue is the initiation point for reverse transcription. In the final DNA‐RNA compound, the portion of the RNA transcribed by reverse transcriptase is eliminated by RNase H. The DNA‐RNA compound is present in several hundred copies per cell. Its biological function is unknown at present.
Molecular cloning and expression of a locus (mdoA) implicated in the biosynthesis of membrane‐derived oligosaccharides in Escherichia coli
Authors: Lacroix J.‐M. , Tempête M. , Menichi B. , Bohin J.‐P. 
Published: October 2006

SummaryMutants of Escherichia coli defective in the mdoA locus are blocked at an early stage in the biosynthesis of membrane‐derived oligosaccharides. The mdoA locus has now been cloned into multicopy plasmids. A 5kb DNA fragment is necessary to complement mdoA mutations. Cells harbouring the mdoA+ plasmid produced three to four times more MDO than wild‐type cells. MDO overproduction did not affect the degree of MDO substitution with sn‐1‐phosphoglycerol residues. The biosynthesis of MDO remained under osmotic control in overproducing strains.
Molecular and genetical analysis of the fructose‐glucose transport system in the cyanobacterium Synechocystis PCC6803
Authors: Zhang C.‐C. , Durand M.‐C. , Jeanjean R. , Joset F. 
Published: October 2006

SummaryComplementation for glucose transport capacity of deficient mutants from Synechocystis PCC6803 allowed the cloning of the corresponding gene, glcP. The protein predicted from one open reading frame (ORF) in the DNA sequence was 468 residues long. It showed 46–60% amino acid sequence homology and similarity in size and predicted structure (including twelve probable membrane‐spanning regions) with a group of non‐phosphorylating sugar transporters from mammals, yeasts and Escherichia coli. A second ORF, 64 base pairs downstream from glcP, was detected. Its function, dispensable under auto‐ and heterotrophic conditions, could not be determined. Genetic analysis of mutants confirmed that the resistance to fructose, acquired simultaneously with the deficiency in glucose transport, resulted from mutations in the glcP gene, whose approximate location could be determined.
Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257
Authors: Rouch D. A. , Messerotti L. J. , Loo L. S. L. , Jackson C. A. , Skurray R. A. 
Published: October 2006

SummaryTrimethoprim resistance mediated by the Staphylococcus aureus multi‐resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789‐790 bp), the outside two of which are flanked by directly repeated 8‐bp target sequences. IS257 has imperfect terminal inverted repeats of 27‐28 bp and encodes for a putative transposase with two potential α‐helix‐turn‐α‐helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram‐negative bacteria and with ISS 1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim‐resistant DHFRs from Gram‐negative bacteria and with chromosomally encoded DHFRs from Gram‐positive and Gram‐negative bacteria. 5’to dfrA is a thymidylate synthetase gene, designated thyE.
Distribution of msDNAs among serotypes of enteropathogenic Escherichia coli strains
Authors: Lim D. , Gomes T. A. T. , Maas W. K. 
Published: October 2006

SummaryA genetic element, called a retron, is present in certain Escherichia coli strains. It consists of genes for the production of a covalently linked DNA‐RNA compound and a reverse transcriptase. The presence of a retron can be detected by testing for a satellite DNA band by polyacrylamide gel electrophoresis. This DNA band consists of the DNA portion of the DNA–RNA compound and is called msDNA (multicopy single‐stranded DNA). In a survey of intestinal E. coli isolates we detected msDNAs in classical enteropathogenic (EPEC) strains and in strains with aggregative adherence to tissue‐culture cells (AA), but not in enteroinvasive (EIEC) and enterotoxigenic (ETEC) strains. Among 76 EPEC strains belonging to 14 different serotypes, msDNA was found to be present in 7 serotypes. In total, five different types of msDNA were found, although within each serotype, the msDNAs were the same. These results suggest that different retrons are clonally inherited.
Involvement of OmpF during reception and translocation steps of colicin N entry
Authors: Bourdineaud J.‐P. , Frerobe H.‐P. , Lazdunski C. , Pagès J.‐M. 
Published: October 2006

Summary[125I]‐colicin N binds to OmpF receptor sites (70000 per cell) with an average Kassoc of 3.2 × 106 M−1 at 23°C. Monoclonal antibody directed against a cell‐surface‐exposed epitope of OmpF is able to compete with the binding of the colicin in vitro and also to protect against colicin N in vivo. OmpF is an absolute requirement for colicin N uptake. OmpC cannot serve as a substitute for OmpF during translocation across the outer membrane under receptor bypass conditions, which is in contrast to colicin A. Colicin N does not cross‐react with various monoclonal antibodies directed against colicin A.
Effects of N‐methyl‐D‐aspartate receptor ligands on yeast sporulation
Authors: Piñón R. 
Published: October 2006

SummaryEarlier studies have suggested that glutamate may play an important role in the transition between the mitotic (vegetative) and meiotic (sporulative) stages of the life cycle in the yeast Saccharomyces cerevisiae. Glutamate is also a major excitatory neurotransmitter in the vertebrate brain, and its actions are mediated by the excitatory amino acid (EAA) family of receptors, the three best‐characterized of which are the N‐methyl‐d‐aspartate (NMDA), quisqualate (Q), and kainate (K) receptors. As an initial test of the possibility that glutamate action in S. cerevisiae might be mediated by an EAA‐like receptor mechanism, the effects of ligands that define the functional domains of the vertebrate NMDA receptor have been examined. The responses of S. cerevisiae cells to ligands that act at four distinct sites on the NMDA receptor provide the first evidence for an NMDA‐like receptor‐mediated system involved in the control of yeast sporulation.
Uncharged tRNA, protein synthesis, and the bacterial stringent response
Authors: Goldman E. , Jakubowski H. 
Published: October 2006

SummaryUncharged tRNA has been shown in vivo to have an active role both in the stringent response, and in modulating the rate of translational elongation. Both of these effects appear to be mediated by codon–anticodon interactions on the ribosome. Although the involvement of uncharged tRNA in the stringent response was expected from in vitro experiments, it has only recently been confirmed in vivo. Inhibition of translation by cognate uncharged tRNA was not expected, and a model is proposed in which excess uncharged tRNA competes with charged tRNA (in ternary complex) for the 30S component of the ribosomal A site. When uncharged tRNA is in sufficient excess over charged tRNA, interaction of uncharged tRNA with the 50S component of the A site occurs as well, leading to a stringent response. The cell has a continuum of responses to decreasing aminoacyl‐tRNA levels: in moderately limited conditions, the proportion of uncharged tRNA increases, and the translation rate is slowed; under more severe limitations, uncharged tRNA provokes a stringent response, with pleiotropic consequences for the cell.
Structure, localization and function of FanF, a minor component of K99 fibrillae of enterotoxigenic Escherichia coii
Authors: Simons B. L. , Willemsen P. T. J. , Bakker D. , Roosendaal B. , De Graaf F. K. , Oudega B. 
Published: October 2006

SummaryThe DNA sequence of the K99 fanF gene, encoding FanF, was determined. An open reading frame of 999bp was found. The primary structure of FanF was deduced and analysis revealed the presence of a signal sequence of 22 amino acid residues. The mature protein contains 311 amino acid residues (Mr 33905 D). The amino acid sequence of FanF showed similarity with the K88ab major subunit FaeG.A specific mouse antiserum against FanF was prepared by constructing and purifying a hybrid CroLacZ‐FanF protein. Minicell analysis, immunoblotting and immunoelectronmicroscopy revealed a pool of FanF in the periplasm of K99‐producing cells and showed, furthermore, that FanF Is a minor component of K99 fibrillae, present at the top and in or along the shaft of the K99 fibrillar structures.A fanF mutant plasmid was constructed. Cells harbouring this plasmid produced all K99‐specific proteins, except FanF, but produced 0.1% of the K99 fibrillae relative to ‘normal’ K99‐producing cells. Electron microscopic observations showed that cells defective in fanF produce only a few (apparently short) K99 fibrillae. FanF, therefore, was supposed to play a role in initiation and elongation of K99 fibrillae formation.Thin‐layer chromatography experiments involving purified receptor material showed that FanF is not required for binding of K99 fibrillae to the ganglioside receptor. Fibrillae produced by an adhesion‐negative strain carrying a mutation in the K99 major fibrillar subunit were shown to contain a normal amount of FanF.
Mapping of the msDNA operon in the chromosome of Escherichia coli B
Authors: Lim D. , Maas W. K. 
Published: October 2006

SummaryAn msDNA operon, consisting of genes for msDNA and a reverse transcriptase, is present in Escherichia coli B and absent from E. coli K12. We have found that the msDNA operon is located on a DNA fragment, longer than 15 kb, that is absent from E. coli K12. Using conjugation, P1 transduction, and nucleic acid hybridization between E. coli B and E. coli K12 strains, we have located the position of the msDNA operon on the E. coli B chromosome at a site that corresponds to minute 19 on the genetic map and to position 900 on the physical map of the E. coli K12 chromosome.
Pro‐subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro
Authors: Ohta Y. , Inouye M. 
Published: October 2006

SummaryThe formation of active subtilisin E from pro‐subtilisin E requires the removal of the N‐terminal pro‐sequence of 77 residues. Pro‐subtilisin E produced in Escherichia coli using a pINIII‐ompA vector was first extracted with 6M guanidine‐HCl and 5M urea and purified to homogeneity in the presence of 5M urea. Upon drop dialysis against 0.2M sodium phosphate buffer (pH 6.2), the purified pro‐subtilisin in 5M urea was processed to active subtilisin of which the N‐terminal sequence and migration in SDS‐polyacrylamide gel electrophoresis were identical to those of authentic active subtilisin E. This process was found to be very sensitive to the ionic strengths and anions used. Under the optimum conditions (dialysis against 0.5 M (NH4)2SO4 and 1 mM CaCl2 in 10mM Tris‐HCl buffer (pH 7.0) at 4°C for 1 h), approximately 20% of pro‐subtitisin E was converted to active subtilisin E. The activation process was not inhibited by Streptomyces subtilisin inhibitor, and pro‐subtiiisin E in which the active site was mutated (Asp32 to Asn) was unable to be processed under the optimum conditions. These results confirmed the previous hypothesis that the processing of pro‐subtilisin occurs by an intramolecular, autoprocessing mechanism.
Transcriptional analysis of the gene encoding pyruvate formate‐lyase‐activating enzyme of Escherichia coli
Authors: Sauter M. , Sawers R. G. 
Published: October 2006

SummaryThe act gene of Escherichia coli encodes the pyruvate formate‐lyase‐activating enzyme which is necessary for the post‐translational modification of pyruvate formate‐lyase. The gene is located 191 bp downstream from the pfl structural gene. Northern blot analysis revealed that the act transcript is monocistronic and that transcription is independent of pfl gene expression. Through mapping of the 5′ and 3′ ends of the act transcript, sequences could be identified showing similarity to both an Escherichia coliσ70 promoter and to a rho‐independent transcription terminator. Expression of the act gene was analysed with the aid of chromosomally integrated transcriptional and translational lacZ fusions. The results verified that the act gene is transcribed from its own promoter and that expression of the gene is essentially constitutive. Anaerobiosis led only to a two‐fold increase in expression over that observed in aerobically grown cells and this elevated expression was independent of the transcriptional regulator, Fnr. Moreover, effectors such as pyruvate and nitrate, which substantially influence anaerobic transcription of the pfl gene, did not affect act gene expression.
Purine biosynthesis in Escherichia coli K12: structure and DNA sequence studies of the purHD locus
Authors: Flannigan K. A. , Hennigan S. H. , Vogelbacker H. H. , Gots J. S. , Smith J. M. 
Published: October 2006

SummaryThe de novo purine biosynthetic enzymes 5‐amino‐4‐imidazolecarboxamide‐ribonucleotide (AICAR) transformylase (EC 2.1.2.3), IMP cyclohydrolase (EC 3.5.4.10) and glycineamide‐ribonucleotide (GAR) synthetase (EC 2.1.2.2) are encoded by the purHD locus of Escherichia coli. The DNA sequence of this locus revealed two open reading frames encoding polypeptides of Mr57335 and 45945 (GAR synthetase), respectively, that formed an operon. The DNA sequence, maxicell and complementation analyses all supported the concept that the Mr 57335 polypeptide is the product of the purH gene and encodes a bifunctional protein containing both AICAR trans‐formylase and IMP cyclohydrolase activities. The 5′ end of the purHD mRNA was determined by primer extension mapping and contains two regions of dyad symmetry capable of forming ‘hairpin’ loops where the formation of the one would prevent the formation of the other but not vice versa. Regulation by the purR gene product was explained by the discovery of a purR binding site in the purHD control region.
The coagulase of Staphylococcus aureus 8325‐4. Sequence analysis and virulence of site‐specific coagulase‐deficient mutants
Authors: Phonimdaeng P. , O'Reilly M. , Nowlan P. , Bramley A. J. , Foster T. J. 
Published: October 2006

SummaryThe sequence of the coagulase gene (coa) from Staphylococcus aureus strain 8325‐4 is reported. The deduced amino acid sequence of the coagulase protein is compared with previously reported sequences of coagulases from strains 213 and BB. The secreted mature forms of coagulase proteins are composed of three distinct segments: (i) the N‐terminal 150–270 residues, which are c. 50% identical, (ii) a central region with high (>90%) residue identities, and (iii) a C‐terminal region composed of repeated 27‐amino‐acid residue sequences. The variable N‐terminal sequences are probably responsible for antigenic differences among coagulases of different serotype. The region of coagulase which binds to prothrombin and activates it to form staphylothrombin is also located in the N‐terminal half of the protein.A site‐specific substitution mutation in the coa gene, which abolished plasma clotting activity, was isolated by recombinational allele‐replacement in strains 8325‐4 and M60. The Coa− mutants did not show diminished virulence in subcutaneous and intramammary infections of mice. No evidence for a role for coagulase in virulence of toxigenic or non‐toxigenic strains was obtained. This contradicts findings of several groups using Coa− mutants generated by chemical mutagenesis and suggests that the earlier results were obtained with strains that had suffered additional mutations in virulence‐related genes.
Activation potassium efflux from Escherichia coli by glutathione metabolites
Authors: Elmore M. J. , Lamb A. J. , Ritchie G. Y. , Douglas R. M. , Munro A. , Gajewska A. , Booth I. R. 
Published: October 2006

SummaryThe mechanism by which N‐ethylmaleimide (NEM) elicits potassium efflux from Escherichia coli has been investigated. The critical factor is the formation of specific glutathione metabolites that activate transport systems encoded by the kefB and kefC gene products. Formation of N‐ethyl‐succinimido‐S‐glutathione (ESG) leads to the activation of potassium efflux via these transport systems. The addition of dithiothreitol and other reducing agents to cells reverses this process by causing the breakdown of ESG and thus removing the activator of the systems. Chlorodinitrobenzene, p‐chloromercuribenzoate and phenylmaleimide provoke similar effects to NEM. Iodoacetate, which leads to the formation of S‐carboxymethyl‐glutathione, does not activate the systems but does prevent the action of NEM. It is concluded that the KefB and KefC systems are gated by glutathione metabolites and that the degree to which they are activated is dependent upon the nature of the substituent on the sulphydryl group.
Comparison and cross‐species expression of the acetyl‐CoA synthetase genes of the ascomycete fungi, Aspergillus nidulans and Neurospora crassa
Authors: Connerton I. F. , Fincham J. R. S. , Sandeman R. A. , Hynes M. J. 
Published: October 2006

SummaryThe genes encoding the acetate‐inducible enzyme acetyl‐coenzyme A synthetase from Neurospora crassa and Aspergillus nidulans (acu‐5 and facA, respectively) have been cloned and their sequences compared. The predicted amino acid sequence of the Aspergillus enzyme has 670 amino acid residues and that of the Neurospora enzyme either 626 or 606 residues, depending upon which of the two possible initiation codons is used. The amino acid sequences following the second alternative AUG show 86% homology between the two species; the extended N‐terminal sequences show no homology. The Neurospora protein is characterized by the appearance of the S(T)PXX sequence motif where the amino acid homologies break down. The codon usage is biased in both genes, with a marked deficiency, especially in Neurospora, of codons with A in the third position.The facA transcribed sequence contains six introns: one in the long leader sequence, one in the 5′ coding sequence not homologous with acu‐5, and four within the sequence that is largely similar to that of acu‐5. Only one intron, corresponding in size and position to the furthest downstream of the facA introns, is found in acu‐5. The evolution of introns during the divergence of these two Ascomycete fungi is discussed. Each of the two genes has been transferred by transformation into the other species. Each species is evidently able to splice out the other's introns. Most transformants have normal acetate‐induction of acetyl‐CoA synthetase, emptying that the two genes respond to transcriptional control signals common to both species, in spite of the striking divergence of their 5′ ends.
The life‐cycle proteins RodA of Escherichia coli and SpoVE of Bacillus subtilis have very similar primary structures
Authors: Joris B. , Dive G. , Henriques A. , Piggot P. J. , Ghuysen J. M. 
Published: October 2006

SummaryComparison of the predicted amino acid sequence of the cell‐cycle RodA protein with the National Research Foundation protein sequence database shows that the 370‐amino‐acid RodA, a protein that is essential for wall elongation in Escherichia coli and maintenance of the rod shape of the cell, is highly analogous, in terms of primary structure, with the Bacillus subtilis SpoVE protein involved in stage V of sporulation.
Structure and dynamics of the colicin E1 channel
Authors: Cramer W. A. , Cohen F. S. , Merrill A. R. , Song H. Y. 
Published: October 2006

SummaryThe toxin‐like and bactericidal colicin E1 molecule is of interest for problems of toxin action, polypeptide translocation across membranes, voltage‐gated channels, and receptor function. Colicin E1 binds to a receptor in the outer membrane and is translocated across the cell envelope to the inner membrane. Import of the colicin channel‐forming domain into the inner membrane involves a translocation‐competent intermediate state and a membrane potential‐dependent movement of one third to one half of the channel peptide into the membrane bilayer. The voltage‐gated channel has a conductance sufficiently large to depolarize the Escherichia coli cytoplasmic membrane. Amino acid residues that affect the channel ion selectivity have been identified by site‐directed mutagenesis. The colicin E1 channel is one of a few membrane proteins whose secondary structures in the membrane, predominantly α‐helix, have been determined by physico‐chemical techniques. Hypotheses for the identity of the trans‐membrane helices, and the mechanism of binding to the membrane, are influenced by the solved crystal structure of the soluble colicin A channel peptide. The protective action of immunity protein is a unique aspect of the colicin problem, and information has been obtained, by genetic techniques, about the probable membrane topography of the imm gene product.
Analysis of the structure‐function relationship of Pseudomonas aeruginosa exotoxin A
Authors: Wick M. J. , Hamood A. N. , Iglewski B. H. 
Published: October 2006

SummaryBiochemical and genetic techniques have provided considerable insight into the structure‐function relationship of one of the ADP‐ribosyl transferases produced by Pseudomonas aeruginosa, exotoxin A. Exotoxin A contains a typical prokaryotic signal sequence which, in combination with the first 30 amino‐terminal amino acids of the mature protein, is sufficient for exotoxin A secretion from P. aeruginosa. Determination of the nucleotide sequence and crystalline structure of this prokaryotic toxin allowed a molecular model to be constructed. The model reveals three structural domains of exotoxin A. Analysis of the identified domains shows that the amino‐terminal domain (domain I) is involved in recognition of eukaryotic target cells. Furthermore, the central domain (domain II) is involved in secretion of exotoxin A into the periplasm of Escherichia coli. Evidence also implicates the role of domain II in translocation of exotoxin A from the eukaryotic vesicle which contains the toxin after it becomes internalized into susceptible eukaryotic cells via receptor‐mediated endocytosis. The carboxy‐terminal portion of exotoxin A (domain III) encodes the enzymatic activity of the molecule. The structure of this domain includes a cleft which is hypothesized to be the catalytic site of the enzyme. Several residues within domain III have been identified as having a direct role in catalysis, while others are hypothesized to play an important structural role.