bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mistranslation induces the heat‐shock response in the yeast Saccharomyces cerevisiae
Authors: Grant C. M. , Firoozan M. , Tuite M. F. 
Published: October 2006

SummaryThe synthesis of heat‐shock proteins can be triggered by a variety of stress‐inducing conditions. Here we show that translational misreading caused by growth in the presence of the aminoglycoside antibiotic paromomycin will induce the heat‐shock response in the yeast Saccharomyces cerevisiae. This was demonstrated (i) by the acquisition of thermotolerance, and (ii) by elevated levels of expression of the heat‐shock protein, hsp70. In addition, transcription of the ubiquitin gene (UBI4) was increased in paromomycin‐grown cells. Control experiments with the protein synthesis inhibitor cycloheximide (which does not induce translational misreading) demonstrated that the response was not due to inhibition of protein synthesis per se. These observations strongly suggest that the synthesis of abnormally high levels of aberrant proteins is the trigger of the heat‐shock response in this simple eukaryote.
Nucleotide sequence and analysis of the N‐terminal coding region of the Spodoptera‐activeδ‐endotoxin gene of Bacillus thuringiensis aizawai 7.29
Authors: Sanchis V. , Lereclus D. , Menou G. , Chaufaux J. , Guo S. , Lecadet M.‐M. 
Published: October 2006

SummaryThe nucleotide sequence of a 2711 bp DNA segment which contains the N‐terminal coding sequence and the 5’flanking region of a crystal protein gene (bta) from Bacillus thuringiensis subsp. aizawai 7.29 has been determined. The coding region encodes an 824 amino‐acid polypeptide corresponding to a carboxy‐terminally truncated δ‐endotoxin specifically active against the cotton leaf worm Spodoptera littoralis. Comparison of the deduced amino acid sequence of the bta gene with that of the 4.5, 5.3 and 6.6 kb classes of lepidopteran‐active β‐endotoxins revealed that the Bta sequence contains a very high level of amino acid substitutions in the N‐terminal part of the protoxin molecule. The substitutions are grouped in several highly variable segments separated by highly conserved regions. These conserved domains are also present in the dipteran‐ and coleopteran‐active δ‐endotoxins. The control region of the bta gene shows considerable DNA identity with the control regions of the other lepidopteran‐active genes. Deletions of the 3’region of the gene were carried out and the toxic fraction of the btaδ‐endotoxin was identified with the N‐terminal half of the molecule.
β‐lactamase as a probe of membrane protein assembly and protein export
Authors: Broome‐Smith J. K. , Tadayyon M. , Zhang Y. 
Published: October 2006

SummaryThe enzyme TEM β‐lactamase constitutes a versatile gene‐fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Celts producing translocated forms of β‐lactamase can be directly selected as ampicillin‐resistant colonies, and consequently a β‐lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic β‐lactamase derivative depends on its level of expression, and therefore a β‐lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane‐bound gene products expressed in E. coli.
HeLa cell invasion by a strain of enteropathogenic Escherichia coli that lacks the O‐antigenic polysaccharide
Authors: Riley L. W. , Junio L. N. , Schoolnik G. K. 
Published: October 2006

SummaryThe interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid‐cured derivative strain was examined. An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the 0‐repeating side chain of the lipopolysaccharide. Under light microscopy, the plasmid‐cured derivative strain B171‐4 was observed to interact with HeLa cells in a pattern distinct from LA. Transmission electron microscopy showed that the bacteria were internalized by HeLa cells. In contrast, strain B171 induced pedestal‐like projections and invaginations of the plasma membrane, but was never completely internalized. A quantitative assay to determine the number of internalized bacteria revealed that strain B171‐4 was internalized at levels 30–70‐fold higher than those of aviruient E. coli strains. Cytochalasin B reduced the levels of internalization of both strain B171‐4 and an entero‐invasive E. coli strain (E11), but did not affect LA by strain B171. These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells. However, a plasmid‐determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell.
Isolation and characterization of the gene encoding a novel, thermostable serine proteinase from the mould Tritirachium album Limber
Authors: Samal B. B. , Karan B. , Boone T. C. , Osslund T. D. , Chen K. K. , Stabinsky Y. 
Published: October 2006

SummaryA number of proteinases are induced and secreted into the culture medium of Tritirachium album Limber when the nitrogen source is limited to exogenous proteins. We have constructed a cDNA library using the polyadenylated RNA isolated during the nutritional induction with bovine serum albumin. A full‐length clone of a gene for a new proteinase (named proteinase R) was identified from this library. This clone contained sequences coding for the 108‐amino‐acid prepro‐leader as well as for the 279‐amino‐acid mature preoteinase. Proteinase R apparently belongs to the subtilisin group of serine proteases that contains disulphide bonds. Homology between proteinase R and proteinase K was found to be about 87% at the nucleotide as well as at the amino acid level. The Brookhaven Protein Data Base co‐ordinate file of proteinase K was used as a template to study the proteinase R substitutions in three‐dimensional space. The majority of the substitutions of proteinase R with respect to proteinase K were found to be on the exterior of the protein model.
Prolongation of the yeast life span by the v‐Ha‐RAS oncogene
Authors: Chen J. B. , Sun J. , Jazwinski S. M. 
Published: October 2006

SummaryThe budding yeast Saccharomyces cerevisiae has a finite life span that is defined by the number of times the cell divides. The patterns of expression of certain genes change in a specific manner during the life span, implying that at least some of the manifestations of the ageing process are subject to gene regulation. It has now been determined that the controlled expression of the RAS oncogene in yeast increases the longevity of this organism, indicating that, conversely, a defined alteration in the activity of a single gene can extend this organism's life span. The results suggest that there is a balance between life‐span extension and growth arrest when RAS is expressed. Inasmuch as the homologues of RAS in yeast function to integrate cell metabolism with the cell cycle, these studies raise the possibility that this integrative function may also apply to the co‐ordination of successive cell cycles during the life span.
Identification of developmentally regulated Giardia lamblia cyst antigens using GCSA‐1, a cyst‐specific monoclonal antibody
Authors: Ward H. D. , Kane A. V. , Ortega‐Barria E. , Keusch G. T. , Pereira M. E. A. 
Published: October 2006

SummaryGCSA‐1, a monoclonal antibody raised against cysts generated in vitro was shown to be Giardia cyst‐specific by immunoblot analysis and immunofluorescence. GCSA‐1 recognized four polypeptides ranging from 29–45kD present in the cyst wall. These antigens appeared within eight hours of exposure of trophozoites to encystation medium and were shown to be synthesized by encysting parasites by means of metabolic labelling with [35S]‐cysteine. Trophozoites were not stained by the antibody. GCSA‐1 also reacted with in vivo cysts obtained from faeces of infected humans, gerbils and mice. These data demonstrate that the determinants recognized by GCSA‐1 are early cyst antigens which are developmentally regulated and conserved components of the cyst wall. The actual role of the antigens detected by GCSA‐1 in encystation are unknown, but they represent a potential target for strategies directed at inhibiting this process.
Expression patterns of genes encoding elastase and controlling mucoidy: co‐ordinate regulation of two virulence factors in Pseudomonas aeruginosa isolates from cystic fibrosis
Authors: Mohr C. D. , Rust L. , Albus A. M. , Iglewski B. H. , Deretic1 V. 
Published: October 2006

SummaryTranscriptional patterns of lasB and algD were compared in isogenic mucoid and non‐mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients. The lasB gene encodes elastase, a major proteolytic enzyme secreted by P. aeruginosa, while algD is required for the synthesis of alginate, an exopolysaccharide frequently overproduced by strains infecting cystic fibrosis patients. A possible coregulation at the transcriptional level of these major virulence determinants was analysed. The lasB and algD genes showed inverse levels of promoter activity. The lasB promoter was active in non‐mucoid cells and inactive in mucoid cells (in four out of five tested pairs), while the algD promoter was active in mucoid cells and silent in non‐mucoid cells in all cases. When PAO568, a model strain for the analysis of control of the alginate system, was grown under conditions promoting mucoidy, the algD promoter was activated, whereas lasB mRNA could not be detected. This effect was reversed when the cells were grown in a medium suppressing mucoidy. Insertional inactivation of algR, a member of the signal‐transduction systems regulating algD transcription, although abolishing algD expression and rendering cells non‐mucoid, did not alter the nature of the induction and repression patterns of lasB seen in the parental strain PAO568. These results suggest that the lasB gene and the alginate system are co‐ordinately regulated at a level parallel to or above the algR gene.
Genetic analysis of the cytochrome c‐aa3 branch of the Bradyrhizobium japonicum respiratory chain
Authors: Bott M. , Bolliger M. , Hennecke H. 
Published: October 2006

SummaryFurther genetic evidence is provided here that Bradyrhizobium japonieum possesses a mitochondria‐like electron‐transport pathway: 2[H]→UQ→bc1→c→aa3→O2. Two Tn5‐induced mutants, COX122 and COX132, having cytochrome c oxidase‐negative phenotypes, were obtained and characterized. Mutant COX122 was defective in a novel gene, named cycM, which was responsible for the synthesis of a c‐type cytochrome with an Mr, of 20000 (20K). This 20K cytochrome c appeared to catalyse electron transport from the cytochrome bc1, complex to the aa3‐type terminal oxidase and, unlike mitochondrial cytochrome c, was membrane‐bound in B. japonicum. The Tn5 insertion of mutant COX132 was localized in coxA, the structural gene for subunit I of cytochrome aa3. This finding also led to the cloning and sequencing of the corresponding wild‐type coxA gene that encoded a 541 ‐amino‐acid protein with a predicted Mr of 59247. The CoxA protein shared about 60% sequence identity with the cytochrome aa3 subunit I of mitochondria. The B. japonicum cycM and coxA mutants were able to fix nitrogen in symbiosis with soybean (Fix+). In contrast, mutants described previously which lacked the bc1 complex did not develop into endosymbiotic bacteroids and were thus Fix−. The data suggest that a symbiosis‐specific respiratory chain exists in B. japonicum in which the electrons branch off at the bc1 complex.
Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitutions in listeriolysin O
Authors: Michel E. , Reich K. A. , Favier R. , Berche P. , Cossart P. 
Published: October 2006

SummaryListeriotysjn O (LLO), a major virulence factor of the intracellular bacterium Listens monocytogenes, shares with other known ‘thiol‐activated toxins’ a conserved undecapeptide, ECTGLAWEWWR, located in the C‐terminal region of the protein and containing the unique cysteine of the molecule. Single amino acid substitutions were created in this region to study the role of cysteine and tryptophan residues in the lytic activity of LLO as well as in the virulence of the bacterium. Transformation of a transposon‐induced non‐haemolytic mutant with plasmids carrying the mutated genes allowed allele exchange and transfer of mutations on to the chromosome by in vivo recombination. The mutant strains secreted a full‐length 59 kilodalton LLO. A decrease of 25% in the haemolytic activity in culture supernatants was observed in the case of mutation Cys‐484 to Ala and of 80% for mutation Cys‐484 to Ser. Mutations Trp‐491 and Trp‐492 to Ala decreased activity by, respectively, 95% and 99.9%. LLOs produced by the mutants, as the wild type, were active at low pH, inhibited by cholesterol, and able to bind to cell membranes. A close relationship was found between virulence of mutants in the mouse model and haemolytic activity in their culture supematants. These results demonstrate that the thiol group of Cys‐484 is not essential for either haemolytic activity in vitro or virulence in vivo. In contrast, Trp‐492 appears to be required for both haemolytic activity and virulence. The finding that the nearly non‐haemolytic mutant Trp‐492‐Ala persisted in the spleen for several days after inoculation indicates that mutagenesis of a virulence determinant can attenuate virulence and provides a novel approach to the development of live vaccine strains.
Functions of the flagellar modes of rotation in bacterial motility and chemotaxis
Authors: Eisenbach M. 
Published: October 2006

SummaryBacteria swim by rotating their flagella, the rotation being due to a motor located at the base of each flagellum. In this paper the correlation between motor function and mode of swimming is reviewed, with special emphasis on recent data that indicate that the motor is a three‐state device. Novel findings with regard to the motor function and bioenergetics are surveyed, and mechanisms are proposed to account for these findings.
Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome
Authors: Médigue C. , Bouché J. P. , Hénaut A. , Danchin A. 
Published: October 2006

SummaryThis paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from the Escherichia coli chromosome on the restriction map established by Kohara et al. (1987). The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Kohara et al. (1987). Therefore, the sequenced genes available in the EMBL library may be localized on the E. coli chromosome restriction map. This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions. Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between the E. coli K12 strains used by the various laboratories involved in DNA sequencing. The program is versatile enough to be used with other genomes.
Mucoid Pseudomonas aeruginosa in cystic fibrosis: mutations in the muc loci affect transcription of the algR and algD genes in response to environmental stimuli
Authors: Deretic V. , Govan J. R. W. , Konyecsni W. M. , Martin D. W. 
Published: October 2006

SummaryIncreased levels of alginate biosynthesis cause mucoidy in Pseudomonas aeruginosa, a virulence factor of particular importance in cystic fibrosis. The algR gene product, which controls transcription of a key alginate biosynthetic gene, algD, is homologous to the activator members of the two‐component, environmentally responsive systems (NtrC, OmpR, PhoB, ArcA, etc). In this report, we show that mutations in the muc loci, (muc‐2, muc‐22, and muc‐23, in the standard genetic P. aeruginosa strain PAO, as well as a mapped muc allele in an isolate from a cystic fibrosis patient) affect transcription of algD and algR. This influence was strongly dependent on environmental factors. Regulation by nitrogen was observed in all strains examined, but the absolute transcriptional levels, determining the mucoid or non‐mucoid status, were strain (muc allele)‐dependent. Increased concentrations of NaCl in the medium, an osmolyte which is elevated in cystic fibrosis lung secretions, resulted in an increased algD transcription and mucoid phenotype in a muc‐2 strain; the same conditions, however, produced a nonmucoid phenotype in the muc‐23 background and abolished algD transcription. Mutations in the muc loci may cause mucoidy by deregulating the normal response of the alginate system to environmental stimuli.
Molecular characterization of the nodulation gene, nodT, from two biovars of Rhizobium leguminosarum
Authors: Surin B. P. , Watson J. M. , Hamilton W. D. O. , Economou A. , Downie J. A. 
Published: October 2006

SummaryDNA sequencing of the nodlJ region from Rhizobium leguminosarum biovar trifolii revealed the nodT gene immediately downstream of nodJ. DNA hybridizations using a nodT‐specific probe showed that nodT is present in several R. leguminosarum strains. Interestingly, a flavonoid‐inducible nodT gene homologue in R. leguminosarum bv. viciae is not in the nodABCIJ operon but is located downstream of nodMN. The sequence of the nodT gene from bv. viciae was determined and a comparison of the predicted aminoacid sequences of the two nodT genes shows them to be conserved; the predicted protein sequences appear to have a potential transit sequence typical of outer‐membrane proteins. Mutations affecting nodT in either biovar had no observed effect on nodulation of the legumes tested.
Export and processing analysis of a fusion between the extracellular heat‐stable enterotoxin and the periplasmic B subunit of the heat‐labile enterotoxin in Escherichia coli
Authors: Guzmán‐Verduzco L.‐M. , Kupersztoch Y. M. 
Published: October 2006

SummaryAs an initial approach in the study of the mechanism of secretion of the extracellular heat‐stable enterotoxin of Escherichia coli (STA), and in order to use this polypeptide as an extracellular carrier we previously constructed a fusion between the complete STA toxin (pre‐pro‐STA) and the mature B subunit of the periplasmic heat‐labile enterotoxin (LTB); the resulting STA‐LTB hybrid was not secreted to the extracellular environment, and cells expressing the hybrid lysed at temperatures above 35°C. In this work we have established that the hybrid is initially detected as pre‐pro‐STA‐LTB and converted to pro‐STA‐LTB, which lacks the 19amino acids that share the properties of a signal peptide; the sequenced 17 amino‐terminal residues of pro‐STA‐LTB defined the processing site of pre‐pro‐STA‐LTB at pro_3phe_2ala_1 ↓ gln+1. This process was sensitive to an energy uncoupler (CCCP) and was correlated with translocation of pro‐STA‐LTB across the inner membrane. Additionally, we are able to show that although pre‐pro‐STA‐LTB is processed at 37°C and 29°C, it is more efficiently processed at the latter temperature. At 37°C, pro‐STA‐LTB was poorly released into the periplasm, resulting in accumulation of this protein, pre‐pro‐STA‐LTB, and pre‐β‐lactamase in the inner membrane, and in cell lysis. In contrast, at 29°C pro‐STA‐LTB was localized in the periplasm and in the inner membrane, and pre‐pro‐STA‐LTB and pre‐β‐lactamase did not accumulate; however, translocation of periplasmic pro‐STA‐LTB across the outer membrane still did not occur, and a second processing step that would eliminate the pro segment from pro‐STA‐LTB was never observed. Thus, the fusion of pre‐pro‐STA and LTB resulted in a polypeptide that, while incompatible with secretion to the extracellular medium, is exported to the periplasm in a temperature‐conditional fashion. This latter observation is consistent with an STA secretion pathway whereby pre‐pro‐STA is first processed to periplasmic pro‐STA by the removal of 19‐amino‐acid signal peptide.
A soluble 60 kilo Dalton antigen of Chlamydia spp. is a homologue of Escherichia coli GroEL
Authors: Bavoil P. , Stephens R. S. , Falkow S. 
Published: October 2006

SummaryTwo major 60kD protein species can be separated by differential detergent extraction in Chlamydia spp. A Sarkosyl‐soluble 60kD protein is (i) structurally and antigenically distinct from the previously characterized 60kD Omp2 outer membrane protein; and (ii) antigenically related to a bacterial common antigen of similar molecular weight which includes a 65 kD myco‐bacterial antigen and the GroEL heat‐shook protein of Escherichia coli. Among GroEL homologues, the chlamydial protein (chl‐GroEL) uniquely displays affinity towards immobilized thiol groups. The significance of this property is discussed with respect to the synthesis and assembly of the chlamydial disulphide‐rich cell wall late in the growth cycle. Chl‐GroEL is identical to the Triton X‐100‐soluble, ocular delayed‐type hypersensitivity agent (Morrison et al., 1989), an essential component in the development of blinding trachoma. An autoimmune mechanism for chronic chlamydial diseases based on chl‐GroEL homology to host proteins is hypothesized.
Transcriptional regulation of the cytR repressor gene of Escherichia coli: autoregulation and positive control by the cAMP/CAP complex
Authors: Gerlach P. , Valentin‐Hansen P. , Bremer E. 
Published: October 2006

SummaryThe Escherichia coli cytR‐encoded repressor protein (CytR) controls the expression of several genes involved in nucleoside and deoxynucleoside uptake and metabolism. The cytR promoter was identified by determining the transcriptional initiation site of the cytR gene. A chromosomal cytR‐lacZ+ operon fusion was isolated and used to study the regulation of cytR. We show that cytR expression is negatively controlled by the CytR protein and positively affected by the cAMP/CAP complex. Foot printing studies with purified CAP protein revealed two CAP binding sites upstream of the cytR promoter. A previousty described mutation (cytR*) in the cloned cytR gene, which results in the phenotypic suppression of a CytR operator mutation in the tsx P2 promoter, was analysed. DN A sequence analysis of the cytR* mutation revealed a G‐C to an A‐T base pair transition at position ‐34 bp relative to the translational initiation site of cytR. This point mutation activates a cryptic promoter that is stronger than the wild‐type cytR promoter and leads to overproduction of the CytR repressor.