bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of pyelonephritis‐associated pili phase‐variation in Escherichia coli: binding of the Papl and the Lrp regulatory proteins is controlled by DNA methylation
Authors: Nou Xiangwu , Skinner Brett , Braaten Bruce , Blyn Lawrence , Hirsch Dwight , Low David 
Published: October 2006

SummaryExpression of pyelonephritis‐associated pili (Pap) in Escherichia coli is under a phase‐variation control mechanism in which individual cells alternate between pili+ (ON) and pili− (OFF) states through a process involving DNA methylation by deoxyadenosine methylase (Dam). Methylation of two GATC sites (GATC1028 and GATC1130) within the pap regulatory region is differentially inhibited in phase ON and phase OFF cells. The GATC1028 site of phase ON cells is non‐methylated and the GATC1130 site is fully methylated. Conversely, in phase OFF cells the GATC1028 site is fully methylated whereas the GATC1130 site is non‐methylated. Two transcriptional activators, Papl and Lrp (leucine‐responsive regulatory protein), are required for this specific methylation inhibition. DNA footprint analysis using non‐methylated pap DNAs indicates that Lrp binds to a region surrounding the GATC1130 site, whereas Papl does not appear to bind to pap regulatory DNA. However, addition of Lrp and Papl together results in an additional DNasel footprint around the GATC1028 site. Moreover, Dam methylation inhibits binding of Lrp/Papl near the GATC1028 site and alters binding of Lrp at the GATC1130 site. Our results support a model in which Dam and Lrp/Papl compete for binding near the GATC1028 site, regulating the methylation state of this GATC site and, consequently, the pap transcription state.
Cloning and characterization of the bundle‐forming pilin gene of enteropathogenic Escherichia coli and its distribution in Salmonella serotypes
Authors: Sohel Indira , Puente Jose Luis , Murray William J. , Vuopio‐Varkila Jaana , Schoolnik Gary K. 
Published: October 2006

Summary bfp, the structural gene of the major repeating bundle‐forming pilus (BFP) subunit, was cloned from the enteroadherent factor (EAF) plasmid of enteropathogenic Escherichia coli (EPEC) strain B171 (0111:NM). The bfp open reading frame encoded a 193‐amlno‐acid protein; comparison of this sequence with the biochemically determined N‐terminal amino acid sequence showed that the mature pilin protein is comprised of 180 amino acids, that this sequence is similar to other members of the type IV pilin family, and that it is preceded by a 13‐amino‐acid signal peptide. Expression of the cloned bfp structural gene in an EPEC strain that had been cured of the EAF plasmid yielded a 21000 dalton protein that co‐migrated with the BFP precursor protein. Thus, other genes, probably carried by the EAF plasmid, are required for the maturation of the bfp product and for the production of extracellular pilus filaments. Use of bfp as a hybridization probe showed that homologous sequences are present in all tested EPEC strains and in 13 of 16 tested Salmonelia serotypes. Fifty per cent of these bfp probe‐sensitive salmonellae exhibited the localized‐adherence (LA) phenotype when incubated with tissue culture cell monolayers, a trait previously associated with EAF plasmid‐containing EPEC strains. Scanning electron micrographs of a bfp probe‐positive, LA‐positive Salmonella dublin strain showed that it grows as adherent colonies on infected monolayers and that within these colonies, BFP‐like fibres form inter‐bacterial linkages. For EAF plasmid‐containing EPEC strains and for severai Salmonella serotypes, BFP expression may lead to the development of adherent colonies on epithelial surfaces early in the infective process.
Identification and characterization of IS 1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family
Authors: Bhugra B. , Dybvig K. 
Published: October 2006

SummaryInsertion sequence (IS) elements are mobile genetic elements found in prokaryotes. We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements. By subcloning a single strain of M. pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event. The nucleotide sequences of the element, designated IS 1138, and the target site into which it inserted were determined. IS1138 consists of 1288bp with 18bp perfect terminal inverted repeats. Sequence analysis of the target site before and after insertion of IS1138 identified a 3bp duplication of target DNA flanking the element. The predicted amino acids encoded by the major open reading frame of IS 1138 share significant similarity with the transposases of the IS3 family. Southern hybridization analysis indicates that repetitive sequences similar to IS 1138 are present in most, if not all, strains of M. pulmonis, but Is1138–like sequences were not detected in other mycoplasmal species.
Characterization of the diphtheria toxin receptor‐binding domain
Authors: Rolf John M. , Eidels Leon 
Published: October 2006

SummaryThe carboxyl‐terminal region of diphtheria toxin (DT) has been analysed in order to determine regions of receptor recognition. Biochemical cleavage of the toxin with hydroxylamine (HA) was used to generate the peptides HA9DT (residues 454–535), HA6DT (residues 482–535), and HA3DT (residues 454–461). Characterization of HA6DT demonstrated that the final 54 amino acids of DT are sufficient to constitute the receptor‐binding domain of the toxin. Within HA9DT, the region encompassing HA3DT and containing the highly cationic polyphosphate‐binding site did not contribute to the binding ability of HA6DT. Consistent with this observation, HA3DT itself did not compete for binding of radiolabelled DT to Vero cells. A 30‐amino acid synthetic peptide composed of residues 506–535 did not block receptor binding of DT, indicating that residues toward the amino‐terminus of HA6DT, or the entire HA6DT region, are required for receptor recognition.
Characterization of mutations in divlB of Bacillus subtilis and cellular localization of the DivlB protein
Authors: Harry E. J. , Stewart B. J. , Wake R. G. 
Published: October 2006

SummaryFour temperature‐sensitive mutations in the divlB gene of Bacillus subtilis have been localized to the region corresponding to the C‐terminal half of the 263‐residue DivlB protein. Antiserum was raised to the 80%C‐terminal portion lying on one side of a putative transmembrane (hydrophobic) segment, and used to examine aspects of the nature and localization of the DivIB protein in the cell. A single DivIB species of a size equal to the full‐length protein encoded by the divIB gene was detected in wild‐type cells. Cell fractionation studies established that DivIB is associated preferentially with the cell envelope (membrane plus cell wall), with approximately 50% being released into solution upon treatment of cells with lysozyme under conditions that yield protoplasts. Of the remaining 50%, approximately half remained firmly associated with the membrane fraction. On the basis of the‘positive‐inside rule’of von Heijne (1986) it is suggested that the topology of membrane‐bound DivlB is such that the long C‐terminal portion is directed to the outside and the smaller N‐terminal portion to the inside of the cell. DivlB in protoplasts was rapidly degraded by proteinase K under conditions where there was no general proteolysis of the cytoplasmic proteins. This is consistent with its absence from the cytoplasm, and with the predicted membrane topology.Septum positioning in a divIB null mutant, which grows as filaments at temperatures of 30°C and below, was found to be normal. It appears that DivlB is needed for achieving the appropriate rate of initiation of septum formation at normal division sites. It is proposed that the C‐terminal portion of DivlB, localized on the exterior surface of the membrane and in juxtaposition to the peptidoglycan, normally interacts with another protein (or proteins) to initiate septum formation.
Homologous overexpression of a light‐driven anion pump in an archaebacterium
Authors: Heymann Jürgen A. W. , Havelka Wendy A. , Oesterhelt Dieter 
Published: October 2006

SummaryThe retinal protein halorhodopsin (HR), a light‐driven chloride pump from Halobacterium halobium, was homologously overexpressed in this archaebacterium. Two DNA expression systems differing in their promoter region were investigated. The halo‐opsin, hop, promoter coupled to the hop gene gave an increased level of HR synthesis. However, the extent of expression was driven by the copy number of the shuttle vector and did not reach the magnitude of the bacterio‐opsin, bop, promoter system. Employing a gene fusion approach, the promoter for the bop gene was used to drive expression of the hop gene. A shuttle vector containing a bop‐hop‐cartridge was transformed into a HR‐deficient strain and blueish‐coloured transformants were obtained. The bop promoter expressed HR to an extent where a specific membrane fraction resembled the crystalline purple membrane of BR in terms of the lipid to protein ratio. HR could, therefore, be easily isolated in a natural membrane‐bound state. This allows for direct use in biophysical studies without the application of deter‐gents. This was the first successful overexpression of a 7‐helical transmembrane protein and may be extended to other proteins of this family.
Regulation of peptide antibiotic production in Bacillus
Authors: Marahier M. A. , Nakano M. M. , Zuber P. 
Published: October 2006

SummaryIn Bacillus species, starvation leads to the activation of a number of processes that affect the ability to survive during periods of nutritional stress. Activities that are induced include the development of genetic competence, sporulation, the synthesis of degradative enzymes, motility, and antibiotic production. The genes that function in these processes are activated during the transition from exponential to stationary phase and are controlled by mechanisms that operate primarily at the level of transcription initiation. One class of genes functions in the synthesis of special metabolites such as the peptide antibiotics tyrocidine and gramicidin S as well as the cyclic lipopeptide surfactin. These genes include the grs and tyc operons in Bacillus brevis, which encode gramicidin S synthetase and tyrocidine synthetase, respectively, and the srfA operon of Bacillus subtilis which encodes the enzymes of the surfactin synthetase complex. Peptide antibiotic biosynthesis genes are regulated by factors as diverse as the early sporulation gene product Spo0A, the transition‐state regulator AbrB, and gene products (ComA, ComP, and ComQ) required for the initiation of the competence developmental pathway.
Strategies in pathogenesis: mechanistic specificity in the detection of generic signals
Authors: Duban M. Eugene , Lee Kyunghee , Lynn David G. 
Published: October 2006

SummaryThe virulence genes of the plant pathogen Agrobacterium tumefaciens are induced by more than 40 low‐molecular‐weight phenolic compounds. The prevailing opinion is that (i) wound‐derived phenols produced on breach of the integrity of the cell wall act as the initiating signal in a series of events which results in host cell transformation, and (ii) a classical membrane receptor, putatively VirA, is responsible for the recognition of all such phenolic inducers. Here, we argue that the discovery of the subset of inducers that are relatives of the dehydrodiconiferyl alcohol glucoside (DCG) growth factors redirects our attention to work on the plant wound as a site of cell division, and suggests that we further explore the implications of early work on the relationship between transformation efficiency and the status of the cell cycle of the host. In addition, we argue that the significant structural diversity allowed in the para position of the phenol ring of inducers suggests that a receptor–ligand interaction based solely on structural recognition is insufficient, but that recognition followed by a specific proton transfer event may be sufficient to explain vir induction activity. Hence, the specificity of the response of A. tumefaciens may be a consequence of the features required for a chemical reaction to occur on the receptor surface. Finally, we review affinity labelling studies which exploit this phenol detection mechanism and which provide evidence that the phenol receptor may be other than VirA, the sensory kinase of the two component regulatory system implicated in Agrobacterium virulence. Taken together, these hypotheses form a model which has predictive and therefore experimental value, and which may be of broader interest in that it calls attention to the possibility of an intricate co‐evolution of signalling pathways of host and pathogen.
In situ hybridization analysis of developmental stages of Pneumocystis carinii that are transcriptionally active for a major surface glycoprotein gene
Authors: Haidaris Patricia J. , Wright Terry W. , Gigliotti Francis , Fallon Margaret A. , Whitbeck April A. , Haidaris Constantine G. 
Published: October 2006

SummaryAn abundant glycoprotein on the surface of Pneumocystis carinii, termed gpA or gp120, is thought to play a role in the interaction of this opportunistic pathogen with its host. Using RNA:RNA hybridization techniques, the in situ expression of gpA mRNA in developmental forms of the organism was investigated in a ferret model of P. carinii pneumonia. The results suggested that the relative abundance of gpA‐specific mRNA was variable in different developmental stages of ferret P. carinii. P. carinii localized along the epithelial lining of alveoli were transcriptionally active. Immunocytochemical detection of gpA and Giemsa staining suggested that many of these organisms were trophic forms of P. carinii. While no detectable gpA mRNA signal was found in the majority of P. carinii cysts, a portion of identifiable cysts co‐localized with significant levels of gpA mRNA signal. Differential staining of the cyst wall with Gomori's methenamine silver suggested that the transcriptionally active P. carinii cysts were the intermediate or precyst forms of the organism, while the cysts with no detectable mRNA signal were either mature or empty (excysted). Alveolar macrophages were observed surrounded by transcriptionally active organisms; however, no gpA‐transcriptional activity was detected within macrophages. Taken together, the results suggest that transcription of gpA occurs in forms of P. carinii that are actively replicating, and in close proximity or contact with, alveolar epithelial cells.
Molecular analysis of a cytotoxin‐converting phage, φCTX, of Pseudomonas aeruginosa: structure of the attP–cos–ctx region and integration into the serine tRNA gene
Authors: Hayashi Tetsuya , Matsumoto Hideki , Ohnishi Makoto , Terawaki Yoshiro 
Published: October 2006

SummaryThe Pseudomonas aeruginosa ctx gene encoding cytotoxin is carried by a temperate phage φCTX. The genome of φCTX is a 35.5 kb double‐stranded DNA with cohesive ends (cos). It is unique in that the ctx gene and attP site of φCTX exist very close to the respective cohesive ends. In this study, we determined the structure of this attP–cos–ctx region. The termini of φCTX are 21‐base 5′ extended‐single‐stranded DNAs. The ctxgene is located 361 bp downstream of the left end (cosL). The attP core sequence of 30 bp exists only 647 bp apart from the right end (cosR). The attP–cos–ctx region contains six kinds of repeats and integration host factor‐binding sequences and showed sequence‐directed static bends, suggesting its potential to form a highly ordered structure. In addition, φCTX was found to integrate into the serine tRNA gene which was mapped to the 43–45 min region on the P. aeruginosachromosome.
Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non‐overlapping functional regions in the encoded protein
Authors: Ter‐Avanesyan Michael D. , Kushnirov Vitaly V. , Dagkesamanskaya Adilya R. , Didichenko Svetlana A. , Chernoff Yury O. , Inge‐Vechtomov Sergey G. , Smirnov Vladimir N. 
Published: October 2006

Summary SUP35is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C‐terminal part similar to the elongation factor EF‐1α and a unique N‐terminal sequence of 253 amino acids. Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence. Functional studies of these deletion mutants showed that: (i) only the EF‐1α‐like C‐terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N‐terminal part of the Sup35 protein or the full‐length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of [psi+] strains; (iii) expression of the C‐terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N‐ or C‐terminal segments of the Sup35 protein can bind to 80S ribosomes. Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions.
The virA promoter is a host‐range determinant in Agrobacterium tumefaciens
Authors: Turk Stefan C. H. J. , Nester Eugene W. , Hooykaas Paul J. J. 
Published: October 2006

SummaryThe limited host range (LHR) Agrobacterium tumefaciens strain Ag162 is an isolate with a narrow host range. Introduction of the wide host range (WHR) virA gene is essential for extending the host range to Kalanchoë daigremontiana. In this report we show that the region upstream of the ATG start codon is responsible for the LHR phenomenon and that this is probably due to the non‐inducibility of the LHR virA promoter. By comparing the characteristics of the LHR and WHR VirA receptor proteins, it was found that the LHR VirA protein is able to activate the WHR VirG protein in the presence of acetosyringone and that this acetosyringone‐dependent vir‐induction is enhanced by the presence of d‐glucose, as in the case of WHR VirA proteins. These results indicate that the domains, acting as receptors for sugars and phenolic signals, must be conserved between the LHR and WHR VirA receptor proteins.
Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length
Authors: Bastin David A. , Stevenson Gordon , Brown Peter K. , Haase Antje , Reeves Peter R. 
Published: October 2006

SummaryWe report the identification and sequence from Escherichia coli and Salmonella enterica strains of the cld gene, encoding the chain‐length determinant (CLD) which confers a modal distribution of chain length on the O‐antigen component of lipopolysaccharide (LPS). The distribution of chain lengths in the absence of this gene fits a model in which as the chain is extended there is a constant probability of 0.165 of transfer of growing chain to LPS core, with termination of chain extension. The data for E. coli 0111 fit a model in which the CLD reduces this probability for short chains and increases it to 0.4 for longer chains, leading to a reduced number of short chain molecules but an increase in numbers of longer molecules and transfer of essentially all molecules by chain length 21. We put forward a model for O‐antigen polymerase which resembles the ribosome and fatty acid synthetase in having two sites, with the growing chain being transferred from a D site onto the new unit at the R site to extend the chain and then back to the D site to repeat the process. It is proposed that the CLD protein and polymerase form a complex which has two states:‘E’facilitating extension and T facilitating transfer to core. The complex is postulated to enter the E state as O‐antigen polymerization starts, and to shift to the T state after a predetermined time, the CLD acting as a molecular clock. The CLD is not O‐antigen or species‐specific but the modal value does depend on the source of the cld gene.
Bacillus subtilis FlhA: a flagellar protein related to a new family of signal‐transducing receptors
Authors: Carpenter Phillip B. , Ordal Georges W. 
Published: October 2006

Summary The Bacillus subtilis flhA gene lies in the major che/fla operon, a transcription unit that spans 26 kilobases (kb) of DNA. flhA encodes a 677‐amino‐acid polypeptide that is a strong candidate for an integral membrane protein. The sequence of FlhA displays substantial homology to a newly identified family of putative signal‐transducing receptors that have been implicated in diverse cellular processes. FlhA is the first member of this family to be described from a Gram‐positive bacterium. We demonstrate that flhA is a flagellar gene and that FlhA is required in trans for the formation of products from some, but not all, B. subtilis motility‐related operons that are regulated by the αD form of RNA polymerase. We suggest that FlhA is a component of a signalling system that is involved with the formation of some flagellar gene products during the biosynthesis of the flagellum.
Structural analysis and demonstration of the 29 kDa antigen of pathogenic Entamoeba histolytica as the major accessible free thiol‐containing surface protein
Authors: Flores Becky M. , Batzer Mark A. , Stein Murry A. , Petersen Carolyn , Diedrich Dana L. , Torian Bruce E. 
Published: October 2006

SummaryThe 29 kDa protein of pathogenic Entamoeba histolytica is a cysteine‐rich surface antigen which we recently characterized by cDNA sequencing and by using monoclonal antibodies which differentiated between pathogenic and non‐pathogenic clinical isolates. To determine the structure and biochemical attributes of this protein, a repertoire of immunologcal techniques using monoclonal antibodies, and radiolabelling were employed. We demonstrated that the 29 kDa protein forms a 60 kDa dimer and a high‐molecular‐mass oligomer(s) on the surface of the organism through disulphide bonds, and is the major accessible free thiol‐containing surface protein of E. histolytica. The deduced amino acid sequence encoding the 29 kDa protein was found to share a common amino acid domain with sequences reported for Helicobacter pylori, Salmonella typhimurium, MER5 gene expressed in murine erythroleukemia cells, Clostridium pasteurianum, and a Bacillus spp.
Cloning and identification of a two‐component signal‐transducing regulatory system from Bacteroides fragilis
Authors: Rasmussen Beth A. , Kovacs Endre 
Published: October 2006

SummaryA DNA fragment was cloned from Bacteroides fragilis that bestowed low‐level tetracycline resistance to Escherichia coli strains harbouring the cloned fragment on a multicopy plasmid. The tetracycline resistance determinant was localized to a 4.3kb BglII–Pstl subfragment of the original clone. DNA sequence analysis of this fragment revealed that it contained an operon encoding two proteins: one of 519 amino acids, RprX, and a second of 236 amino acids, RprY. Protein sequence analysis revealed that the two proteins shared sequence identity with a family of multicomponent signal‐transducing regulatory proteins identified from many diverse bacterial genera. RprX shared identity with the first component of the regulatory system, the histidine protein kinase receptor (for example EnvZ, PhoR, CheA, and VirA). RprY shared identity with the second member of the regulatory protein pair, the regulatory response protein (for example OmpR, PhoB, CheY, and VirG). Expression of these proteins from a multicopy plasmid vector in E. coli resulted in a decrease in the level of the outer membrane porin protein OmpF and an increase in the level of the outer membrane porin protein OmpC. The decrease in OmpF levels correlates with, and may be the cause of, the increased tetracycline resistance. Regulation of the levels of OmpF and OmpC is normally controlled by a multicomponent signal‐transducing regulatory pair of proteins, EnvZ and OmpR. The effect RprX and RprY have on OmpF expression is mediated at the level of transcription. Thus, RprX and RprY may be interfering with the normal regulation of OmpF by OmpR and EnvZ.