bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of the fibronectin‐binding components of Streptococcus pyogenes in Escherichia coli demonstrates that they are proteins
Authors: Talay S. R. , Ehrenfeld E. , Chhatwal G. S. , Timmis K. N. 
Published: October 2006

SummaryThe fibronectin‐binding components (fbcs) of two clinical isolates and a culture collection strain of Streptococcus pyogenes have been analysed. Western immunoblotting of bacterial lysates which had been fractionated on polyacrylamide gels revealed trypsin‐sensitive fibronectin‐binding species. The genes specifying the fbcs were cloned from all three strains and expressed in Escherichia coli using a λ EMBL3 vector. An fbc gene from the culture collection strain was subcloned and expressed in the E. coli expression vector pJLA601, and subjected to deletion analysis. The fibronectin‐binding domain was thereby localized within a 40 kDa truncated peptide encoded by the 1000 bp C‐terminal region of the gene. Southern hybridization experiments demonstrated that the analysed gene was present in the parental S. pyogenes chromosome, but not in the DNA of fbc expressing λ clones obtained from the two clinical isolates. Further evidence for the existence of at least two different types of fbcs in group A streptococci was provided by Western blot analysis of recombinant phage tysates which revealed a complex series of fibronectin‐binding species ranging from 120 to 200 kDa in size and showing strain‐dependent variation in their patterns. As was the case with parental streptococcal strains all of the recombinant fbcs were protease‐sensitive, and treatment with trypsin or pronase resulted in a total loss of fibronectin‐binding activity. Competitive inhibition experiments indicated that lipoteichoic acid was not a significant fbc in the tested streptococcal strains. The biological separation of the principal fbcs of S. pyogenes from lipoteichoic acid, through the cloning and expression of their determinants in the non‐lipoteichoic acid‐producing organism E. coli K‐12, provides unequivocal proof of the protein nature of these components.
Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII In assembly of remaining subunits
Authors: Calder K. M. , McEwen J. E. 
Published: October 2006

SummaryCytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits. Subunits I, II and III are products of mitochondrial genes, while sub‐units IV, V, VI, VII, Vila and VIII are products of nuclear genes. To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized. The strain lacked cytochrome c oxidase activity and haem a/a3 spectra. The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain. The amounts of remaining cytochrome c oxidase sub‐units in the cox7 mutant were examined. Accumulation of subunit I, which is the product of the mito‐chondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products. Results of pulse‐chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I. The synthesis, stability or mitochondrial localization of the remaining nuclear‐encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII. To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial‐encoded subunits I, II and HI with the nuclear‐encoded subunit IV was investigated. Relative to the parental COX7+ strain, the cox7 mutant strain showed no association of sub‐unit IV with subunit I, and decreased association of subunits IV and II. These results provide direct evidence for a role for subunit VII in assembly between mitochondrial‐encoded and nuclear‐encoded cytochrome c oxidase subunits.
Secretion of a potential virulence factor, a fungal ribonucleotoxin, during human aspergillosis infections
Authors: Lamy B. , Moutaouakil M. , Latge J.‐P. , Davies J. 
Published: October 2006

SummaryWe show by cloning and nucleotide sequence analysis that the 18kDa antigen found in the urine of patients suffering from aspergillosis is related to the fungal protein toxins restrictocin and mitogillin. These are inhibitors of translation which act by catalytic inactivation of eukaryotic ribosomes; they may be implicated in the pathogenesis of the disease.
Tn7: a target site‐specific transposon
Authors: Craig N. L. 
Published: October 2006

SummaryThe bacterial transposon Tn7 is an unusual mobile DNA segment. Most transposable elements move at low‐frequency and display little target site‐selectivity. By contrast, Tn7 inserts at high‐frequency into a single specific site in the chromosomes of many bacteria. In the absence of this specific site, called attTn7 in Escherichia coil where Tn7 has been most extensively studied, Tn7 transposes at low‐frequency and inserts into many different sites.Much has recently been learned about Tn7 transposition from both genetic and biochemical studies. The Tn7 recombination machinery is elaborate and includes a large number of Tn7‐encoded proteins, probably host‐encoded proteins and also rather large cis‐acting transposition sequences at the transposon termini and at the target site. Dissection of the Tn7 transposition mechanism has revealed that the DNA strand breakage and joining reactions that underlie the translocation of Tn7 have several unusual features.
Structure and function of pneumolysin, the multifunctional, thiol‐activated toxin of Streptococcus pneumoniae
Authors: Boulnois G. J. , Paton J. C. , Mitchell T. J. , Andrew P. W. 
Published: October 2006

SummaryPneumolysin is a thiol‐activated membrane‐damaging, multifunctional toxin and a known virulence factor of Streptococcus penumoniae. The toxin can interfere with the functioning of both cellular and soluble components of the human immune system which protects against penumococcal infection. Different amino acids within the toxin which are important in promoting oligomerization of the toxin in membranes and for the generation of functional lesions have been identified by site‐directed mutagenesis. Pneumolysin can also activate the classical pathway of complement, and this appears to involve antibody binding (via Fc) by a region of the toxin homologous to C‐reactive protein, a human acute‐phase protein also capable of classical pathway activation and implicated in host defence against pneumococcal infection.
Microbial pathogenesis and tyrosine dephosphorylation: surprising‘bedfellows'
Authors: Clemens J. C. , Guan K. , Bliska J. B. , Falkow S. , Dixon J. E. 
Published: October 2006

SummaryThe importance of parasite‐directed phosphatases in such diseases as smallpox and the bubonic plague emphasizes the need to understand the molecular events associated with normal function of protein tyrosine phosphatase in eukaryotic cells.
The kis and kid genes of the parD maintenance system of plasmid R1 form an operon that is autoregulated at the level of transcription by the co‐ordinated action of the Kis and Kid proteins
Authors: Ruiz‐Echevarria. M. J. , Berzal‐Herranz A. , Gerdes K. , Diaz‐Orejas R. 
Published: October 2006

SummaryStability mediated by the parD system of plasmid R1 is modulated by a killer protein, Kid, and by an antagonist of this function, Kis. Determination of the 5′ ends of ParD transcripts, revealed that the genes coding for these proteins are transcribed from a single promoter. Analysis of the 3′ end of the ParD RNAs indicated the existence of two transcripts: one of them coding for the Kis and Kid proteins, and the other coding only for Kis. Analysis of the effects of parD+ and kis‐ recombinants on the β‐galactosidase levels expressed by different transcriptional and translational parD‐lacZfusions, and on the ParD RNA levels determined by a derepressed parD mutant, indicated that the Kis and Kid proteins repress co‐ordinately the parD system at the transcriptional level. We discuss the relevance of these results in terms of the activities of the Kis and Kid proteins and in the context of the stabilization mediated by parD.
An Agrobacterium two‐component regulatory system for the detection of chemicals released from plant wounds
Authors: Winans S. C. 
Published: October 2006

SummaryCrown gall tumorigenesis by Agrobacterium tumefaciens requires the co‐ordinate transcriptional induction of a set of pathogenesis genes. At least three classes of environmental stimuli act synergistically to induce these genes, (i) monocyclic aromatic hydrocarbons such as acetosyringone, coniferyl alcohol, and vanillin, (ii) neutral or acidic monosaccharides such as glucose and glucuronic acid, and (iii) acidic pH. Three proteins are required to sense and respond to these stimuli: (i) VirA, a transmembrane sensory protein and histidine protein kinase. (ii) VirG, a transcriptional activator which is phosphorylated by phosphoryl VirA, and (iii) ChvE, a periplasmic sugar‐binding protein. VirA and VirG are members of the so‐called two‐component family of regulatory proteins. This regulatory system continues to offer new discoveries in the areas of signal transduction. host‐microbe interactions, and host range.
Characterization of a plasmid from Helicobacter pylori encoding a replication protein common to plasmids in Gram‐positive bacteria
Authors: Kleanthous H. , Clayton C. L. , Tabaqchali S. 
Published: October 2006

SummaryA 1.5kb cryptic plasmid was isolated from Helicobacter pylori. Low‐stringency hybridization analysis using this plasmid as a DNA probe revealed base sequence homology with other plasmids in this species. Nucleotide sequence analysis identified an open reading frame encoding a putative polypeptide of 25 kDa. This protein showed marked amino acid sequence similarity to replication‐initiation proteins commonly found in small plasmids endogenous to Gram‐positive bacteria which replicate by the ‘rolling‐circle’ mechanism. Sequence motifs corresponding to the origins‐of‐replication consensus sequences were found on this cryptic plasmid. DNA and oligonucleotide probes to these plasmid replication sequences were used in hybridization analysis to identify similar sequences in other H. pylori plasmids. We believe this is the first plasmid isolated from a Gram‐negative bacterium to show replication determinants characteristic of the ‘rolling‐circle’ group of plasmids from Gram‐positive bacteria. The cloned plasmid will be used to develop a shuttle‐vector for H. pylori.
Site‐specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis
Authors: Martin C. , Mazodier P. , Mediola M. V. , Gicquel B. , Smokvina T. , Thompson C. J. , Davies J. 
Published: October 2006

SummaryA method which allowed the stable integration of DNA fragments at a single site (attB) in the chromosome of Mycobacterium smegmatis was developed using an integrative element from Streptomyces ambofaciens, pSAM2. Vectors containing an Escherichia coli replicon (pBR322), the kanamycin resistance gene from Tn903 for selection in mycobacteria, and a fragment of pSAM2 containing the int gene as well as the attachment site (attP) were constructed and introduced to M. smegmatis by electroporation. Transformants showed stable integration of the plasmid into a single site (attB) of the mycobacterial genome. This approach should be valuable for analyses of gene expression in various mycobacterial species and permit the development of stable recombinant mycobacterial vaccine strains expressing bacterial or viral genes inserted in pSAM2.
An O antigen can interfere with the function of the Yersinia pseudotuberculosis invasin protein
Authors: Voorhis D. L. , Dillon S. , Formal S. B. , Isberg R. R. 
Published: October 2006

Summary Escherichia coli strains harbouring the Yersinia pseudotuberculosis inv gene are able to enter cultured mammalial cells. We show here that this property is not shared by all enteric bacteria, since Shigella flexneri 2a cured of its virulence‐associated plasmid and harbouring the inv gene is unable to enter mammalian cells efficiently. Mapping studies showed that the region of the chromosome responsible for this phenotype includes rfaB, a locus involved in the production of O antigen. S. flexneri 2a strains that express O antigen were unable to enter mammalian cells, even though invasin was efficiently expressed and localized, showing that this structure interferes with invasin activity. The O antigen either masks invasin or sterically hinders the ability of the mammalian cell receptor to bind this protein.
Role of transcription pausing in the control of the pyrE attenuator in Escherichia coli
Authors: Andersen J. T. , Jensen K. F. , Poulsen P. 
Published: October 2006

SummaryExpression of the Escherichia coli pyrE gene is regulated by transcription attenuation in the intercistronic orfE–pyrE region and modulated by the distance between the transcribing RNA polymerase and the leading ribosome as a function of the supply of UTP and GTP. In this communication we show that pyrE expression is hyper‐repressed in vivo following addition of uracil in strains carrying the nusAcs10 mutation. This phenotype, previously seen in rpsL 1204 strains whose ribosomes are pseudodependent on streptomycin and work at suboptimal elongation rate, indicates that RNA polymerase escapes from the ribosomes in the pyrE attenuator region in the nusA mutant. In vitro transcription studies revealed that the build‐up of the full‐length attenuated orfE transcript occurred more slowly in the presence of the NusA protein than in its absence. Moreover, the NusA protein enhanced several transcription pauses through the orfE gene. These effects were more pronounced when low concentrations of either UTP or GTP were used than at low concentrations of either CTP or ATP. The results indicate that the NusA protein is required for proper regulation of pyrE gene expression and is involved, together with the NTP pools, in maintaining the coupling between transcription and translation in the pyrE attenuator region by inhibiting RNA chain elongation.
The products of Rhizobium genes, psi and pss, which affect exopolysaccharide production, are associated with the bacterial cell surface
Authors: Latchford J. W. , Borthakur D. , Johnston A. W. B. 
Published: October 2006

SummaryTranslational fusions between a mutant phoA (lacking its promoter, ribosomal binding site and signal peptide sequence) and Rhizobium‘symbiotic’ genes were isolated. Since these fusions expressed alkaline phosphatase (AP), the product of phoA, the genes into which phoA was inserted apparently specify proteins located in the bacterial periplasm or cell membrane, the compartment in which AP has activity. These genes were psiA and genes upstream of psiA (psiA is required for normal nodule development and strains with multicopy psiA fall to make exopolysaccharide (EPS) and to nodulate). Fusions between phoA and pss (exo) genes, which are required for EPS production, also resulted in the expression of AP indicating that products of these pss genes were located at the cell surface. Using grus fusions to psiA and pssA, we found that the former was expressed in N2‐fixing bean root nodules but the latter was not.
Pseudomonas aeruginosa LasB mutant constructed by insertional mutagenesis reveais elastolytic activity due to alkaline proteinase and the LasA fragment
Authors: Wolz C. , Hellstern E. , Haug M. , Galloway D. R. , Vasil M. L. , Döring G. 
Published: October 2006

SummaryThe extracellularly secreted endopeptidase elastase (LasB) is regarded as an important virulence factor of Pseudomonas aeruginosa. It has also been implicated in the processing of LasA which enhances elastolytic activity of LasB. In order to Investigate the role of LasB in virulence and LasA processing, a LasB‐negative mutant, PA01E, was constructed by insertional mutagenesis of the LasB structural gene, lasB, in P. aeruginosa PAO. An Internal 636 bp lasB fragment of the plasmid pRB1803 was ligated into a derivative of the mobilization vector pSUP201–1. The resulting plasmid, pBRMOB‐LasB, was transformed into Escherichia coli and transferred by filter matings to the LasB‐positive P. aeruginosa strain, PA01. Plasmid integration in the lasB site of the chromosome was confirmed by Southern blot analysis. Radioimmunoassay and immunoblotting of PA01E supernatant fluids yielded no detectable LasB (<1 ng ml‐1 LasB). The absence of LasB in PA01E was further proven by the inability of its culture supernatant fluid to cleave transferrin or rabbit immunogiobulin G (IgG) after a 72 h incubation. The residual proteolytic activity of PA01E culture supernatant fluid was attributed to alkaline proteinase (Apr), since it was totally inhibited by specific antibodies against Apr. Residual elastolytic activity in culture supernatant fluid of PAO1E was due to the LasA fragment and to the combined action of the LasA fragment with Apr on elastin. The sizes of purified LasA from PA01 and PA01E were identical (22 kDa). These results show that, besides LasB and the LasA fragment, Apr may also act on elastin in the presence of the LasA fragment and that the proteolytic processing of LasA in P. aeruginosa is independent of LasB.
Binding of basement‐membrane laminin by Escherichia coli
Authors: Valkonen K. H. , Veijola J. , Dagberg B. , Uhlin B. E. 
Published: October 2006

SummaryAn invasive Escherichia coli (EIEC) isolate was found to bind basement‐membrane laminin in a saturable and time‐dependent manner. Excess of unlabelled laminin inhibited the binding of the radioactively labelled protein. Non‐invasive E. coli K‐12 exhibited only low‐level laminin binding but introduction of the virulence‐associated plasmid from the EIEC isolate led to high‐level binding. Expression of a receptor for laminin on the bacteria was therefore associated with the presence of the virulence plasmid. Scatchard plot analysis Indicated approximately 1000 receptors per bacterial cell, and a Kd of high‐affinity binding of 0.5 pM. A laminin‐binding protein which correlated with the presence of the plasmid was isolated and characterized. Its sequence of the eight amino‐terminal amino acids was identical to that of the LamB protein of E. coli, although the molecular mass of the two in sodium dodecyl sulphate/polyacrylamide gel (SDS‐PAGE) appeared to be slightly different. Both proteins reacted in immunoblot assays with polyclonal antisera raised against either protein, and both proteins bound laminin. Southern‐blot hybridization analysis established that both the EIEC strain and the K‐12 strains with or without the virulence plasmid contained one lamB gene only, and no laminin‐binding protein appeared when the virulence plasmid was introduced into bacteria deleted for the lamB gene. On the basis of these results we suggest that native LamB protein of E. coli or a modified variant of it serves as a major receptor for laminin binding and is present at an increased level in invasive E. coli containing the virulence plasmid.
Nucleotide sequence of the ipaBCD structural genes of Shigella dysenteriae
Authors: Yao R. , Palchaudhuri S. 
Published: October 2006

SummaryA 9kb EcoRI and two PstI fragments from the virulence plasmid of Shigella dysenteriae CG097 were shown to contain all ipa genes by probing with Shigella flexneri ipaB, ‐C, ‐D and ‐A gene probes. The DNA sequences of S. dysenteriae ipaBC genes were very similar to those of S. flexneri M90T and S. flexneri YSH6000, but ipaD differed by 22 codons from that of S. flexneri. The differences in ipaD may account for the different in vitro host specificities shown by S. dysenteriae and S. flexneri. The nucleotide composition of ipa genes revealed an unusually large number of codons that are rarely used in Escherichia coli chromosomal genes, indicating a different origin.
Cloning of a sequence of Aquaspirillum magnetotacticum that complements the aroD gene of Escherichia coli
Authors: Berson A. E. , Hudson D. V. , Waleh N. S. 
Published: October 2006

SummaryA 2 kb DNA fragment isolated from a cosmid library of Aquaspirillum magnetotacticum strain MS‐1 complements the aromatic‐metabolite requirements and iron‐uptake deficiencies of Escherichia coli and Salmonella typhimurium strains that lack a functional aroD (biosynthetic dehydrodquinase) sequence. All recombinant cosmids selected for their aroD complementation property carry this sequence. No DNA sequence homology has, however, been detected by Southern hybridization between the cloned fragment and the aroD gene of E. coli or the qa2 (catabolic dehydroquinase) gene of Neurospora crassa.
Isolation and characterization of sporulation‐specific promoters in the yeast Saccharomyces cerevisiae
Authors: Coe J. G. S. , Murray L. E. , Kennedy C. J. , Dawes L W. 
Published: October 2006

SummaryA library of random yeast genomic DNA:lacZ fusions has been constructed using an episomal yeast‐Escherichia coli shuttle vector (pCS1). Plasmid pCS1 requires insertion of a promoter and an inframe ATG codon upstream of its resident truncated lacZ gene to regulate expression in yeast. Yeast genomic DNA fragments of 4–6 kb were generated by partial digestion with Sau3A and ligated into the unique BamHI site of plasmid pCS1 to generate a library of 5 × 104 individual E. coli transformants. This library was screened to identify promoter‐lacZ fusions that were expressed uniquely during sporulation. Of 342 yeast transformants that exhibited β‐galactosidase activity, two were found to express the lacZ gene in a sporulation‐specific manner.This paper presents the characterization of two genomic yeast DNA fragments containing promoters that control lacZ expression during the sporulation process. Expression from the promoter present in plasmid pJC18 occurred from 11–21 hours into the sporulation process, while the promoter in plasmid pJC217 was active from 4–14 hours. Staining of nuclear DNA to correlate nuclear morphology with timing of gene expression showed when each of these promoters was active in terms of the morphological stages of sporulation.
A metal‐binding motif implicated in RNA recognition by an aminoacyl‐tRNA synthetase and by a retroviral gene product
Authors: Miller W. Todd , Schimmel Paul 
Published: October 2006

SummaryA randomly generated mutation in Escherichia coli alanine tRNA synthetase compensates for a mutation in its cognate tRNA. The enzyme's mutation occurs next to a Cys‐X2‐Cys‐X6‐His‐X2‐His metal‐binding motif that is distinct from the zinc finger motif found in some DNA‐binding proteins. Instead, the synthe‐tase's metal binding domain resembles the Cys‐X2‐Cys‐X4‐His‐X4‐Cys metal‐binding domain of the gag gene product of retroviruses. For Ala‐tRNA synthetase, the metal bound at the Cys His motif is important specifically for the tRNA‐dependent step of catalysis, and the enzyme‐tRNA interaction is dependent on the geometry of metal co‐ordiNatlon to the enzyme. These data, and the demonstrated sensitivity of RNA packaging to mutations in the metal‐binding domain of the gag gene product of retroviruses, suggest that an aminoacyl‐tRNA synthetase and retroviruses have adopted a related metal‐binding motif for RNA recognition.