bluuebunny/crossref_metadata_2025_split huggingface dataset:
Plasminogen, absorbed by Escherichia coli expressing curli or by Salmonella enteritidis expressing thin aggregative fimbriae, can be activated by simultaneously captured tissue‐type plasminogen activator (t‐PA)
Authors: Sjöbring Ulf , Pohl Gunnar , Olsén Arne 
Published: October 2006

SummaryCurli are fimbrial structures expressed by Escherichia coli that specifically interact with matrix proteins such as fibronectin and laminin. Similar structures are also expressed by Salmonella enteritidis and have been denoted thin aggregative fimbriae. Bacteria expressing curli and thin aggregative fimbriae were found to bind radiolabelled plasminogen as well as the tissue‐type plasminogen activator (t‐PA). By contrast, E. coli carrying a gene locus with an insertionally inactivated chromosomal curlin subunit were unable to bind the two human proteins. The purified subunit polypeptides of curli and thin aggregative fimbriae bound plasminogen and t‐PA with high affinity (1 × 108 to 2 × 108 M‐1). The binding of plasminogen and t‐PA to curli‐expressing E. coli was only partially inhibited by fibronectin and laminin. Plasminogen absorbed from human plasma by curli‐expressing E. coli was readily converted to plasmin by t‐PA; both plasmin and t‐PA were functionally active when bound to the bacteria. A simultaneous binding of fibrinolytic proteins and matrix proteins to fimbriae of E. coli and S. enteritidis could provide these pathogens with both adhesive and invasive properties.
The mRNA for the 23S rRNA methylase encoded by the ermE gene of Saccharopolyspora erythraea is translated in the absence of a conventional ribosome‐binding site
Authors: Bibb Mervyn J. , White Janet , Ward Judith M. , Janssen Gary R. 
Published: October 2006

SummaryTranscriptional analysis of the ermE gene of Saccharopolyspora erythraea, which confers resistance to erythromycin by N6‐dimethylation of 23S rRNA and which is expressed from two promoters, ermEp1 and ermEp2, revealed a complex regulatory region in which transcription is initiated in a divergent and overlapping manner. Two promoters (eryC1p1 and eryC1p2) were identified for the divergently transcribed erythromycin biosynthetic gene eryC1, which plays a role in the formation of desosamine or its attachment to the macrolide ring. Transcription from eryC1p2 starts at the same position as that of ermEp1, but on the opposite strand of the DNA helix, suggesting co‐ordinate regulation of genes for erythromycin production and resistance. ErmEp1 initiates transcription at, and one nucleotide before, the ermE translational start codon. Site‐directed and deletion mutagenesis, combined with immunochemical analysis, demonstrated that the ermEp1 transcript is translated in the absence of a conventional ribosome‐binding site to give rise to the full‐length 23S rRNA methylase. Deletion of the ‐35 region of ermEp1 reduced, but did not abolish, promoter activity, reminiscent of the‘extended ‐10’class of bacterial promoters which, like ermEp1, possess TGN motifs immediately upstream of their 10 regions and which initiate transcription seven nucleotides downstream of the ‐10 region.
Identification of genes involved in the sequestration of iron in mycobacteria: the ferric exochelin biosynthetic and uptake pathways
Authors: Fiss Ellen H. , Yu Shengwei , Jacobs William R. 
Published: October 2006

SummaryMycobacteria produce two siderophores, mycobactin and exochelin. Mycobacterium smegmatis mutants defective in the production of exochelin were isolated using agar medium containing chrome azural S for the sensitive detection of siderophores. Cosmids of genomic libraries from M. smegmatis and Mycobacterium bovis BCG were screened for complementation of the mutation. Subcloning of the complementing M. smegmatis cosmid identified a 4.3 kb fragment required for restoring exochelin biosynthesis. Sequencing of the DNA revealed four open reading frames whose genes were named fxuA. fxuB, fxuC, and fxbA. FxuA, FxuB, and FxuC share amino acid sequence homology with the iron permeases FepG, FepC. and FepD from Escherichia coli. respectively. Deletion analysis identified fxbA as the gene required to restore exochelin biosynthesis in our mutant. Although fxbA does not share amino acid sequence homology with any of the published sequences for siderophore biosynthetic genes, it does show limited homology with the phosphoribosyl‐glycineamide formyltransferases (GAR enzymes) and methionyl‐tRNA formyltransferase over a limited region of the sequence, suggesting that fxbA may code for an enzyme which adds a formyl group in the synthesis of exochelin. A fusion of fxbA with the E. coli lacZ gene demonstrated regulation of gene expression by iron. The ability of M. smegmatis mutants to produce mycobactin in the absence of exochelin supports the hypothesis that exochelin is not a precursor of mycobactin and suggests that the siderophores have independent biosynthetic pathways. In addition, complementation of the M.
Translocation of a hybrid YopE‐adenylate cyclase from Yersinia enterocolitica into HeLa cells
Authors: Sory Marie‐Paule , Cornelis Guy R. 
Published: October 2006

SummaryPathogenic bacteria of the genus Yersinia release in vitro a set of antihost proteins called Yops. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis transfers YopE across the host cell plasma membrane. To facilitate the study of this translocation process, we constructed a recombinant Yersinia enterocolitica strain producing YopE fused to a reporter enzyme. As a reporter, we selected the calmodulin‐dependent adenylate cyclase of Borde‐tella pertussis and we monitored the accumulation of cyclic AMP (cAMP). Since bacteria do not produce calmodulin, cyclase activity marks the presence of hybrid enzyme in the cytoplasmic compartment of the eukaryotic cell. Infection of a monolayer of HeLa cells by the recombinant Y. enterocolitica strain led to a significant increase of cAMP. This phenomenon was dependent not only on the integrity of the Yop secretion pathway but also on the presence of YopB and/or YopD. It also required the presence of the adhesin YadA at the bacterial surface. In contrast, the phenomenon was not affected by cytochalasin D, indicating that internalization of the bacteria themselves was not required for the translocation process. Our results demonstrate that Y. enterocolitica is able to transfer hybrid proteins into eukaryotic cells. This system can be used not only to study the mechanism of YopE translocation but also the fate of the other Yops or even of proteins secreted by other bacterial pathogens.
Evolution of the chaperonin families (HSP60, HSP 10 and TCP‐1) of proteins and the origin of eukaryotic cells
Authors: Gupta Radhey S. 
Published: October 2006

SummaryThe members of the 10 kDa and 60 kDa heat‐shock chaperonin proteins (Hsp10 and Hsp60 or Cpn10 and Cpn60), which form an operon in bacteria, are present in all eubacteria and eukaryotic ceil organelles such as mitochondria and chloroplasts. In archaebacteria and eukaryotic cell cytosol, no close homologues of Hsp10 or Hsp60 have been identified. However, these species (or ceil compartments) contain the Tcp‐1 family of proteins (distant homologues of Hsp60). Phylogenetic analysis based on global alignments of Hsp60 and Hsp10 sequences presented here provide some evidence regarding the evolution of mitochondria from a member of the α‐subdivision of Gram‐negative bacteria and chloroplasts from cyanobacterial species, respectively. This inference is strengthened by the presence of sequence signatures that are uniquely shared between Hsp60 homologues from α‐purple bacteria and mitochondria on one hand, and the chloroplasts and cyanobacterial hsp60s on the other. Within the α‐purple subdivision, species such as Rickettsia and Ehrlichia, which live intracellularly within eukaryotic cells, are indicated to be the closest relatives of mitochondrial Homologues, In the Hsp60 evolutionary tree, rooted using the Tcp‐1 homologue, the order of branching of the major groups was as follows: Gram‐positive bacteria — cyanobacteria and chloroplasts — chlamydiae and spirochaetes —β and γ‐Gram‐negative purple bacteria —α‐purple bacteria — mitochondria. A similar branching order was observed independently in the Hsp10 tree. Multiple Hsp60 homologues, when present in a group of species, were found to be clustered together in the trees, indicating that they evolved by independent gene‐duplication events. This review also considers in detail the evolutionary relationship between Hsp50 and Tcp‐1 families of proteins based on two different models (viz. archaebacterial and chimeric) for the origin of eukaryotic cell nucleus. Some predictions of the chimeric model are also discussed.
Bacterial transposases and retroviral integrases
Authors: Polard P. , Chandler M. 
Published: October 2006

SummaryTransposable genetic elements have adopted two major strategies for their displacement from one site to another within and between genomes. One involves passage through an RNA intermediate prior to synthesis of a DNA copy while the other is limited uniquely to DNA intermediates. For both types of element, recombination reactions involved in integration are carried out by element‐specific enzymes. These are called transposases in the case of DNA elements and integrases in the case of the best‐characterized RNA elements, the retroviruses and retrotransposons. In spite of major differences between these two transposition strategies, one step in the process, that of insertion, appears to be chemically identical. Current evidence suggests that the similarities in integration mechanism are reflected in amino acid sequence similarities between the integrases and many transposases. These similarities are particularly marked in a region which is thought to form part of the active site, namely the DDE motif. In the light of these relationships, we attempt here to compare mechanistic aspects of retroviral integration with transposition of DNA elements and to summarize current understanding of the functional organization of integrases and transposases.
Candida albicans ALS1: domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif
Authors: Hoyer L. L. , Scherer S. , Shatzman A. R. , Livi G. P. 
Published: October 2006

SummaryTransfer of budding Candida albicans yeast cells from the rich, complex medium YEPD to the defined tissue culture medium RPMl 1640 (RPMI) at 37°C and 5% CO2 causes rapid onset of hyphal induction. Among the genes induced under these conditions are hyphal‐specific genes as well as genes expressed in response to changes in temperature, CO2 and specific media components. A cDNA library constructed from cells incubated for 20 min in RPMI was differentially screened with yeast (YEPD)‐ and hyphal (RPMI)‐specific probes resuming in identification of a gene expressed in response to culture conditions but not regulated by the yeast‐hyphal transition. The deduced gene product displays significant identity to Saccharomyces cerevisiaeα‐agglutinin, encoded by AGα1, an adhesion glycoprotein that mediates mating of haploid cells. The presence of this gene in C albicans is curious since the organism has not been observed to undergo meiosis. We designate the C. albicans gene ALS1 (for agglutinin‐like sequence). While the N‐ and C‐termini of the predicted 1260‐amino‐acid ALS1 protein resemble those of the 650‐amino‐acid AGα1, ALS1 contains a central domain of tandem repeats consisting of a highly conserved 36‐amino‐acid sequence not present in AGb1. These repeats are also present on the nucleotide level as a highly conserved 108bp motif. Southern and Northern blot analyses indicate a family of C. albicans genes that contain the tandem repeat motif; at least one gene in addition to ALS1 is expressed under conditions similar to those for ALS1 expression. Genomic Southern blots from several C. albicans isolates indicate that the number of copies of the tandem repeat element in ALS1 differs across strains and. In some cases, between ALS1 alleles in the same strain, suggesting a strain‐dependent variability in ALS1 protein size. Potential roles for the ALS1 protein are discussed.
Molecular characterization of an Aeromonas salmonicida mutant with altered surface morphology and increased systemic virulence
Authors: Noonan Brian , Trust Trevor J. 
Published: October 2006

SummaryThe asoA gene of Aeromonas salmonicida is located approximately 7 kb downstream of the A‐layer structural gene, vapA. A 6 kb Bam HI fragment containing aso A was cloned and marker‐exchange mutagenesis using a kanamycin‐resistance cassette was performed to generate an aso A mutation in the low‐virulence strain A449L. When analysed by electron microscopy, the mutant A449L‐MB exhibited an altered surface morphology. Strands and blebs of membranous material were observed protruding from the disorganized cell surface. This material was shown to contain lipopolysaccharide and A‐layer subunit protein. The disorganization of the surface of A449L‐IV1B had no apparent effect on virulence when the bacteria were administered to rainbow trout (Oncorhynchus mykiss) by bath Immersion. However, when administered by intraperitoneal injection, the mutant A449L‐MB was found to exhibit significantly increased virulence. The predicted amino acid sequence of AsoA shows homology to a number of polytopic membrane proteins involved in translocation across the cytoplasmic membrane.
Reversibility of DnaA protein activity in the‘irreversible’dnaA204 mutant of Escherichia coli
Authors: Hansen Flemming G. , Atlung Tove 
Published: October 2006

SummaryThe dnaA204 mutant, one of the so‐called irreversible dnaA mutants which cannot reinitiate chromosome replication upon a shift from non‐permissive to permissive growth temperature in the absence of protein synthesis, was reinvestigated using flow cytometry and marker frequency analysis. In a temperature downshift experiment and in the presence of protein synthesis the dnaA204 mutant reinitiates chromosome replication very fast. Using a lac promoter‐controlled wild type or a dnaA204 mutant gene carried on a plasmid, we have observed instantaneous initiation of replication when synthesis of DnaA protein is induced in the dnaA204 mutant at 42δC. The data indicate that the dnaA204 mutant after a shift to 42δC still contains functional DnaA protein, but that the activity level is below the initiation threshold. Thus, after synthesis of very small amounts of additional DnaA protein, initiation occurs very fast both after a shift to 30δC, and after induction of DnaA protein synthesis at 42 C. A model describing the processing of DnaA protein in mutants and in the wild type Is presented.
The hrpRS locus of Pseudomonas syringae pv. phaseolicola constitutes a complex regulatory unit
Authors: Grimm C. , Aufsatz W. , Panopoulos N. J. 
Published: October 2006

SummaryThe right part of the hrp cluster of Pseudomonas syringae pv. phaseolicola contains two regulatory genes, the previously described hrpS gene and an adjacent locus, hrpR. In this study we determined the sequence of hrpR and analysed the functional organization of the two genes. HrpR and HrpS show high sequence similarities to each other and to other response regulators of the two‐component regulatory system. This has recently also been described for the hrpRS system of the closely related pathogen Pseudomonas syringae pv. syringae. The results of our genetic analyses strongly indicate that hrpS expression is regulated by the hrpR gene product. DNA‐protein binding studies and site‐directed mutagenesis of the hrpR sequence provided further evidence that HrpR activates hrpS transcription by binding to an activator site. This HrpR binding site has mapped in a fragment which is located 378–609 nucleotides upstream of the hrpS transcription start site. The hrpS transcription start site maps 179 nucleotides upstream of the initiation codon ATG, as determined by primer extension analysis, and is preceded by a typical ‐12/‐24 promoter motif.
Expression of the ATP‐dependent deoxyribonuclease of Bacillus subtilis is under competence‐mediated control
Authors: Haijema Bert Jan , Hamoen Leendert Willem , Kooistra Jan , Venema Gerard , van Sinderen Douwe 
Published: October 2006

SummaryTranscription of the ATP‐dependent deoxynuclease operon (addAB), as monitored by means of an addAB–lacZ transcriptional fusion, has a low, constitutive level and is initiated from a σA type promoter. Transcription of addAB is independent of DNA‐damaging agents known to induce the SOS response in Bacillus subtilis. However, addAB transcription increased significantly during competence development. This competence‐specific induction was dependent on the gene products of srfA., degU and comK, but not on that of recA. Deletion analysis of the addAB promoter region demonstrated that the competence‐specific transcription induction requires DNA sequences located upstream of the addAB promoter that associated with ComK, the competence transcription factor. The latter finding indicates that a direct regulatory link exists between the establishment of the competent state and the synthesis of AddAB, required for recombination of internalized donor DNA.
Missense mutations in the catalase‐peroxidase gene, katG, are associated with isoniazid resistance in Mycobacterium tuberculosis
Authors: Heym Beate , Alzari Pedro M. , Honore Nadine , Cole Stewart T. 
Published: October 2006

SummaryThe toxicity of the powerful anti‐tuberculosis drug isoniazid (INH) is believed to be mediated by the haem‐containing enzyme catalase‐peroxidase, encoded by the katG gene of Mycobacterium tuberculosis. Compelling evidence for this was obtained by studying a panel of INH‐resistant clinical isolates using a novel strategy based on the polymerase chain reaction and single‐strand‐conformation polymorphism analysis (PCR‐SSCP) to detect mutations in katG. In most cases INH resistance was associated with missense mutations while in a small number of strains the gene had been completely, or partially, deleted. The missense mutations fell into two groups, the larger of which contained several independent mutations that affected the N‐terminal peroxidase domain of the protein, resulting in the production of a catalase peroxidase with strongly reduced enzyme activity and increased heat lability. The effects of these substitutions could be interpreted by means of molecular modelling using the crystal structure of the related enzyme cytochrome c peroxidase from yeast as a template. The second group comprises a frequently occurring amino acid substitution and a single mutation that are both located in the C‐terminal domain but do not noticeably alter either enzyme activity or heat stability.
Evidence for a ppGpp‐binding site on Escherichia coli RNA polymerase: proximity relationship with the rifampicin‐binding domain
Authors: Reddy Padmalatha S. , Raghavan Arvind , Chatterji Dipankar 
Published: October 2006

SummaryOn amino acid starvation, Escherichia coli cells exhibit an adaptive facility termed the stringent response. This is characterized by the production of high levels of a regulatory nucleotide, ppGpp, and concomitant curtailment in rRNA synthesis. Various studies reported earlier indicated that RNA polymerase is the site of action of ppGpp although a direct demonstration of the interaction of ppGpp with E. coli RNA polymerase is still lacking. Here we report the labelling of PpGpp with a fluorescent probe, 1‐aminonapthalene‐5‐sulphonate (AmNS), at the terminal phosphates. AmNS‐ppGpp responded much like a ppGpp molecule in an in vitro total transcription assay at selective promoters. Fluorescence titration of the tryptophan emission of RNA polymerase by AmNS‐ppGpp indicated a unique binding site in the absence of template DNA. Competition experiments showed that unlabelled ppGpp binds to the enzyme at the same site. Sigma factor seems to have no effect on this binding. The titration profile is also characterized by a single slope in the Scatchard analysis. The presence of GTP or GDP does not influence the binding of AmNS‐ppGpp with RNA polymerase. Forster's distance measurement was carried out which placed AmNS‐ppGpp 27Å away from the rifampicin‐binding domain of RNA polymerase.
Gonococcal rfaF mutants express Rd2 chemotype LPS and do not enter epithelial host cells
Authors: Schwan E. Thomas , Robertson Brian D. , Brade Helmut , van Putten Jos P. M. 
Published: October 2006

SummaryWe have investigated the function of the lsi‐1 gene of Neisseria gonorrhoeae previously implicated in lipopolysaccharide (LPS)‐inner‐core biosynthesis (Petricoin et al., 1991). Disruption of the gene in gonococcal strain MS11 resulted in the production of LPS that migrated faster than that from an isogenic galE mutant, typical for a mutation that influences the inner‐core region. Complementation of a panel of Salmonella typhimurium mutants with defined defects in rfa loci demonstrated conclusively that the lsi‐1 gene of MS11 is functionally homologous to the rfaF gene, which encodes heptosyltransferase II in both E. coli and S. typhimurium. Comparison of deduced amino acid sequences of the gonococcal and the Salmonella RfaF demonstrated 70% similarity, including 47% identical amino acid residues. Immunochemical analysis of the LPS using monoclonal antibodies directed against chemically defined inner‐core glycoconjugates revealed that the gonococcal and Salmonella Rd2‐Chemotypes were antigenically similar, further extending the genetic and functional homology. Infection experiments in vitro demonstrated that the lsi‐1 mutant could not invade human Chang epithelial cells despite expression of a genetically defined invasion‐promoting gonococcal opacity protein. These data imply that the LPS phenotype is a critical factor for gonococcal invasiveness.
A checkpoint involving RTP, the replication terminator protein, arrests replication downstream of the origin during the Stringent Response in Bacillus subtilis
Authors: Levine Alain , Autret Sabine , Séeror Simone J. 
Published: October 2006

SummaryRegulation of DNA replication in Bacillus subtilis involves a post‐initiation mechanism which is subject to control by the Stringent System, an essential regulatory network, mediated by the alarmone, ppGpp. In detailed studies using DNA‐DNA hybridization procedures, we have now shown that, following the induction of the Stringent Response, replication is blocked downstream of the origin, on the left, close to the hut marker (‐175 kb) and on the right, beyond the soft10 marker (+199 kb). In addition, we provide evidence that inhibition of replication under these conditions requires the replication terminator protein (RTP). In a mutant lacking RTP, a protein normally involved in termination of chromosomal replication through recognition of specific terminator sequences, replication continues past the sites normally blocked by the Stringent Response. These data strengthen the argument that this second level of control of DNA replication occurs at specific sites, the Stringent Terminus (STer) sites, either side of oriC Such sites are presumably related to the sequence involved in RTP recognition at the terminus, terC. We propose that the binding of RTP must be modulated, perhaps through the action of ppGpp, to recognize post‐initiation control sequences during the Stringent Response, in order to block replisome movement. This, therefore, acts as a checkpoint in chromosome elongation.