bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of the periplasm of Neisseria gonorrhoeae
Authors: Judd Ralph C. , Porcella Stephen F. 
Published: October 2006

SummaryThe periplasm of Neisseria gonorrhoeae should be similar to other Gram‐negative bacteria, but no published reports confirm this assumption. We used a periplasmic isolation procedure developed in Escherichia coli to release the periplasmic contents of N. gonorrhoeae. The resultant periplasmic extract lacked lipopolysaccharide, protein markers of inner or outer membranes, surface‐radiolabelled protein components, or ribosomal proteins. The periplasmic extract contained a single haem protein believed to be a c‐type cytochrome known to exist in the periplasm of other Gram‐negative species, and retained significant alkaline phosphatase activity. The dominant protein species released in the periplasmic extract was the gonococcal homologue of elongation factor Tu, a major component released in similar periplasmic extracts of E. coli. These data showed that the extraction procedure selectively released periplasmic components and that the gonococcal periplasm was comparable to that of E. coli. Further analysis of the gonococcal periplasm may provide important insights into the physiology of this pathogen of humans.
Transcriptional organization of the Escherichia coli pilus adhesin K99
Authors: Inoue Olivia J. , Lee John H. , Isaacson Richard E. 
Published: October 2006

SummaryThe production of the Escherichia coli pilus adhesin K99 requires the expression of eight unique proteins: FanA‐H. The transcriptional organization of the K99 operon was investigated by Northern blot analysis. Four RNAs of 0.54, 1.4, 2.5 and 3.5kb were detected. When a fanC probe was used all four RNAs were detected while the use of fanD, fanF and fanG probes detected two RNAs each. Using several deletion and TnphoA insertion mutants it was concluded that there were seven unique K99‐specific transcripts, several of which were of the same approximate sizes (1.4 and 2.5 kb). It also was concluded that K99 was comprised of at least three complementation groups, two of which were regulated by catabolite repression.
Structural variation in the O‐specific polysaccharides ofKlebsiella pneumoniaeserotype O1 and O8 lipopolysaccharide: evidence for clonal diversity inrfbgenes
Authors: Kelly Robert F. , Severn Wayne B. , Richards James C. , Perry Malcolm B. , MacLean Leann L. , Tomás Juan M. , Merino Susana , Whitfield Chris 
Published: October 2006

SummaryThe O‐polysaccharide fraction of the lipopolysaccharide fromKlebsiella pneumoniaeserotype O8 was found to comprise two galactose‐containing homopolymers. Structural analysis, using chemical and high‐field nuclear magnetic resonance (NMR) techniques, established that theK. pneumoniaeO8 polysaccharides are composed of the linear, disaccharide repeating unitsOAc12/6→3)‐β‐d‐Galf‐(1 →3)‐α‐d‐Galp‐(1→d‐Galactan I‐OAc→3)‐α‐d‐Galp‐(1 →3)‐β‐d‐Galp‐(1→d‐Galactan II.K. pneumoniaeO8 mutant RFK‐1 was isolated by resistance to phage KO1‐2; strain RFK‐1 expressed onlyd‐galactan I‐OAc. The1H‐ and13C‐NMR resonances from this O‐polysaccharide indicate that all of theO‐acetyl groups within theK. pneumoniaeO8 polysaccharide are carried ond‐galactan I andO‐acetylation occurs only on the β‐d‐galactofuranose residues; 60% of the available β‐d‐galactofuranose residues are non‐acetylated. TheO‐acetylation of the remaining residues is equally distributed between the O‐2 and O‐6 positions. The carbohydrate backbone structures in the O8 polysaccharide are identical tod‐galactan I and II expressed byK. pneumoniaeO1, accounting for the antigenic cross‐reaction between strains belonging to serotypes O1 and O8. However, the O1 polysaccharides are not acetylated and theO‐acetyl groups present in theK. pneumoniaeserotype O8 polysaccharides provide a structural basis for their recognition as distinct serotypes. Therfb(O‐polysaccharide biosynthesis) gene cluster ofK. pneumoniaeserotype O1 determines the synthesis ofd‐galactan I.rfbKpo1‐specific gene probes were used to examine conservation in therfbgene clusters of otherK. pneumoniaeserotypes which produced‐galactan I. Six O1 strains were examined and all showed hybridization withrfbKpO1probes under conditions of high stringency. Three serotype O2 strains produced‐galactan I and these strains also contained DNA sequences recognized byrfbKpO1probes under high stringency. The physical maps of these homologousrfbchromosomal regions showed some polymorphism. Surprisingly, therfbKpO8region fromK. pneumoniaeserotype O8 was only recognized byrfbKpO1probes under low‐stringency hybridization conditions, providing evidence for two substantially different clonal groups ofrfbgenes fromK. pneumoniaestrains with structurally related O‐antigens.
Activation and mechanism of Clostridium septicum alpha toxin
Authors: Ballard J. , Sokolov Y. , Yuan W.‐L. , Kagan B. L. , Tweten R. K. 
Published: October 2006

Summary Clostridium septicum produces a single lethal factor, alpha toxin (AT), which is a cytolytic protein with a molecular mass of approximately 48kDa. The 48kDa toxin was found to be an inactive protoxin (ATpro) which could be activated via a carboxy‐terminal cleavage with trypsin. The cleavage site was located approximately 4kDa from the carboxy‐terminus. Proteolytically activated ATpro had a specific activity of approximately 1.5 × 106 haemolytic units mg‐1. The trypsin‐activated toxin (ATact) was haemolytic, stimulated a prelytic release of potassium ions from erythrocytes which was followed by haemoglobin release, induced channel formation in planar membranes and aggregated into a complex of Mr >210000 on erythrocyte membranes. ATpro did not exhibit these properties. ATact formed pores with a diameter of at least 1.3‐1.6 nm. We suggest that pore formation on target cell membranes is responsible for the cytolytic activity of alpha toxin.
Chromosomal supercoiling in Escherichia coli
Authors: Miller† William G. , Simons Robert W. 
Published: October 2006

SummaryThe Escherichia coli chromosome is compacted into 40‐50 negatively supercoiled domains. It has been proposed that these domains differ in superhelical density. Here, we present evidence that this is probably not the case. A modified Tn10 transposable element was inserted at a number of locations around the E. coli chromosome. This element, mTn10‐plac‐lacZ+, contains the lac operon promoter, plac, whose activity increases with increasing superhelical density, fused to a lacZ+ reporter gene. Although mTn10‐plac‐lacZ+ fusion expression varies as much as approximately threefold at different insertion sites, the relative levels of expression from these elements are unaffected by replacing plac with the gyrA promoter, pgyrA, which has a reciprocal response to changes in superhelical density. Importantly, topoisomerase mutations and coumermycin, which inhibits DNA gyrase activity, alter mTn10‐plac‐lacZ+ and mTn10‐pgyrA‐lacZ+ fusion expression in expected ways, showing that the elements remain responsive to supercoiling and that topoisomerase activity is required for maintaining superhelical density. Fusion expression is not affected by anaerobic growth or osmotic shock, two physiological conditions thought to alter supercoiling. The approximately threefold difference in mTn10‐plac‐lacZ‐ and mTn10‐pgyrA‐lacZ+ fusion expression observed at different sites may be explained by regional differences in chromosomal copy number that arise from bidirectional replication. Together, these results strongly suggest that the E. coli chromosomal domains do not differ in functional superhelical density.
Novel determinant (comA) essential for natural transformation competence in Neisseria gonorrhoeae and the effect of a comA defect on pilin variation
Authors: Facius Dirk , Meyer Thomas F. 
Published: October 2006

SummaryA novel genetic determinant (comA) has been identified and found to be required for the transformation of piliated Neisseria gonorrhoeae. Mutants in comA of strain MS11 grow normally and are DNA‐uptake proficient but blocked in the translocation of DNA into the cytoplasm. Here we show by site‐specific mutagenesis and genetic complementation that only one of two open reading frames identified in comA is essential for competence: it encodes a protein (ComA) with a predicted size of 74kDa. The comAgene maps upstream of the iga locus and is transcribed in the opposite orientation, probably under the control of a putative σ;54 type promoter. While DNA probes specific for the N. gonorrhoeae iga locus reveal only a little cross‐reactivity with commensal Neisseria species, the neighbouring comA gene appears to be present in most of them. ComA fusion proteins were obtained by in vitro translation. The synthesized gene products migrated atypically in SDS gels indicating its strong hydrophobicity. Several transmembrane α‐helices were predicted from the amino acid sequence of ComA which, in the context of an observed sequence similarity with other inner membrane proteins, suggests a location for the protein in the inner membrane. Using piliated and non‐piliated comA mutants the consequences of transformation deficiency on pilin phase variation were assessed. We show that the comA defect affects some but not all types of DNA rearrangements associated with pilE variation. The results are in agreement with previous observations supporting the notion that multiple recombination pathways contribute to the variability of pilE.
Loss of activity in the secreted form of Escherichia coli haemolysin caused by an rfaP lesion in core lipopolysaccharide assembly
Authors: Stanley Peter L. D. , Diaz Pilar , Bailey Marc J. A. , Gygi Daniel , Juarez Antonio , Hughes Coiin 
Published: October 2006

SummaryA transposon mutant of Escherichia coli 5K was isolated which reduced 10‐ to 50‐fold the secreted extracellular haemolytic activity of cells carrying the complete hlyCABD operon while leaving unaffected the intracellular haemolytic activity and the levels of intracellular and extracellular haemolysin protein, HlyA. The transposon insertion was identified within the rfaP gene (required for attachment of phosphate‐containing substituents to the lipopolysaccharide inner core), and extracellular haemolytic activity was restored in trans by the intact rfaP gene. The toss in cytolytic activity of the secreted HlyA protein was not related to the HlyC‐directed acylation of the protoxin. Activity of the secreted toxin was restored by chaotropic agents and during rate‐zonal centrifugation the mutant‐secreted HlyA migrated as a larger species than the wild type. The results indicate that the rfaP mutation affects the aggregation behaviour of the active toxin during or following the signal peptide‐independent secretion process.
Mode and origin of replication of pSAM2, a conjugative integrating element of Streptomyces ambofaciens
Authors: Hagège Juliette , Pernodet Jean‐Luc , Mann Annick Fried , Guérineau Michel 
Published: October 2006

SummarypSAM2 is an 11 kb integrating element from Streptomyces ambofaciens that is capable of replication. It generates single‐stranded DNA during replication, and is therefore the first Streptomyces integrating element to be described that may belong to the family of elements, called the ssDNA elements, that replicate by a rolling‐circle mechanism. The direction of replication has been identified. The plus origin (ori) of replication and minus origin (M‐O) have been located. Streptomyces lividans harbouring replicating pSAM2 also contain numerous small covalently closed circular DNA molecules (scm) derived from pSAM2. These scm contain ori and extend on both sides of the putative nick site. Sequences at the junction points of these scm are heterogeneous but short direct repeats were always found in the vicinity of these junctions.
Construction of chimeric proteins from the σ;N‐associated transcriptional activators VnfA and AnfA of Azotobacter vinelndii shows that the determinants of promoter specificity lie outside the‘recognition’helix of the HTH motif in the C‐terminal domain
Authors: Jacob Jansen , Drummond Martin 
Published: October 2006

SummaryFunctional chimeras have been generated from the transcriptional activators VnfA and AnfA, which control expression of the alternative nitrogenases in Azotobacter vinelandii. The activation profiles of the native and chimeric proteins have been determined using lacZ fusions to A. vinelandii anf and vnf promoters in Klebsiella pneumoniae. Repiacing the C‐terminal domain of AnfA with that of VnfA gives a protein with the promoter specificity of VnfA, confirming that the C‐terminal domain contains the determinants of promoter specificity. However, substituting the VnfA sequence from the turn in the helix‐turn‐helix motif to the C‐terminus does not alter the promoter specificity of AnfA. These changes in promoter specificity were reflected in changes in affinity for a VnfA‐binding site, as measured by an in vivo repression assay using a lacZ fusion to a synthetic promoter. This supports the assumption that promoter recognition is determined by activator binding to enhancer‐like sequences, and shows that the principal determinants of specific DNA‐binding lie outside the‘recognition’helix. This may be a general feature of transcriptional activators dependent on σ;N (σ54). The chimera with the promoter specificity of VnfA retained the dependence on nitrogenase Fe protein characteristic of AnfA, indicating that this property is not related to particular promoter sequences, but is a function of the central or N‐terminal domains of AnfA.
In a class of its own — the RNA polymerase sigma factor σ;54 (σN)
Authors: Merrick M. J. 
Published: October 2006

SummaryBacteria synthesize a number of different sigma factors which allow the co‐ordinate expression of groups of genes owing to the ability of sigma to confer promoter‐specific transcription initiation on RNA polymerase. In nearly all cases these sigmas belong to a single family of proteins which appear to be related structurally and functionally to the major Escherichia coli sigma factor, σ;70. A clear exception is the Sigma factor σ;54 (σN), encoded by rpoN, which represents a second family of sigmas that is widely distributed in prokaryotes. Studies of σ;54 (σN) have demonstrated that this sigma is quite distinct both structurally and functionally from the σ;70 family and the mode of transcription initiation which it mediates may have more in common with that found in eukaryotes than that which occurs with σ;70 and its relatives.
Crystalline bacterial cell surface layers
Authors: Sleytr Uwe B. , Messner Paul , Pum Dietmar , Sára Margit 
Published: October 2006

SummaryCrystalline arrays of proteinaceous subunits forming surface layers (S‐layers) are one of the most commonly observed prokaryotic cell envelope structures. They are ubiquitous amongst Gram‐positive and Gram‐negative archaeobacteria and eubacteria and, if present, account for the major protein species produced by the cells. S‐layers can provide organisms with a selection advantage by providing various functions including protective coats, molecular sieves, ion traps and structures involved in cell surface interactions. S‐layers were identified as contributing to virulence when present as a structural component of pathogens. In Gram‐negative archaeobacteria they are involved in determining cell shape and cell division. The crystalline arrays reveal a broad‐application potential in biotechnology, vaccine development and molecular nanotechnology.
Construction and use of integrative vectors to express foreign genes in mycobacteria
Authors: Dellagostin Odir A. , Wall Sue , Norman Elizabeth , O'Shaughnessy Tina , Dale Jeremy W. , McFadden JohnJoe 
Published: October 2006

SummaryWe have constructed a mycobacterial integrative vector by placing two copies of the insertion sequence IS900 flanking a kanamycin‐resistance gene into a 'suicide’vector unable to replicate in mycobacteria. The Mycobacterium leprae gene encoding the M. leprae 18kDa protein was cloned between the two copies of IS900 to provide expression signals. Constructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistance. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analysed by Southern blotting. One to five copies of the vector were detected in each transformant. The SIV gag p27 gene and the foot‐and‐mouth disease virus VP1 140‐160 epitope were successfully cloned into the 18kDa gene and expression in M. smegmatis was obtained.
A novel ompC mutation of Escherichia coli K‐12 that reduces OmpC and OmpF levels in the outer membrane
Authors: Misra Rajeev 
Published: October 2006

SummaryA novel ompC mutation was isolated that not only lowered the amount of its own product, OmpC27, but also reduced the level of OmpF present in the outer membrane. ompC27 codes for a mutant OmpC protein that contains two non‐native cysteine residues. The ompC27 allele confers phage resistance by lowering the level of OmpC present in the outer membrane. This effect on OmpC27 was manifested at the level of assembly as a result of disulphide bond formation between the two cysteine residues. This disulphide bonding in OmpC27 also produced a novel phenotype by specifically influencing OmpF levels. The effect of OmpC27 on OmpF was partly a result of a lowering of ompF transcription, and partly a result of an effect at the post‐transcription level. The transcriptional effect is likely to be brought about by a defective membrane as a result of the insertion of the disulphide bond containing OmpC27. The post‐transcriptional effect of OmpC27 on OmpF could be due to interference at the assembly level. In a dsbA::kan1 background where the in vivo disulphide bonding ability was dramatically reduced, the OmpC27‐mediated effects were also curtailed.
Protein F: an adhesin of Streptococcus pyogenes binds fibronectin via two distinct domains
Authors: Sela Shiomo , Aviv Adi , Tovi Aviva , Burstein Israel , Caparon Michael G. , Hanski Emanuel 
Published: October 2006

SummaryThe binding of Streptococcus pyogenes to fibronectin (FN) enables the adherence of this pathogen to target epithelial cells, which is the first necessary step for initiation of infection. Binding is mediated by a bacterial surface protein termed protein F. Here we provide the complete structure of protein F and identify two domains responsible for binding to fibronectin. The first domain is located towards the C‐terminal end of the molecule and is composed of five repeats of 37 amino acids that are completely repeated four times and a fifth time partially. The second domain is adjacent to the first domain and is located on the /V‐terminal side of it. It is composed of a single stretch of 43 amino acids. Protein F expressed in Escherichia coli completely blocked the binding of fibronectin to S. pyogenes. However, mutant proteins that contained only one or the other of the two domains were only capable of partial blockage of binding. Complete blockage of binding of fibronectin could be achieved when a protein extract containing the N‐terminal domain was mixed in a binding reaction with a protein extract containing the C‐terminal domain. Similarly, a purified recombinant protein containing the two domains only, blocked the binding completely. In contrast, a purified recombinant protein containing just the C‐terminal domain, blocked the binding partially. A clone exclusively expressing the C‐terminal domain, completely blocked the binding of the 30 kDa N‐terminal fragment of fibronectin to S. pyogenes, whereas a clone expressing the N‐terminal domain failed to block the binding of this FN fragment. Thus, the two FN‐binding domains of protein F are necessary for maximal bacterial binding and act in concert to enhance the binding to fibronectin. The possibility that the two domains bind to two different regions on the fibronectin molecule is discussed.
A physical and genetic map of the Stigmatella aurantiaca DW4/3.1 chromosome
Authors: Neumann Björn , Pospiech Andreas , Schairer Hans Ulrich 
Published: October 2006

SummaryA physical map of the myxobacterium Stigmatella aurantiaca DW4/3.1 chromosome was constructed by pulsed‐field gel (PFG) long‐range mapping. One‐and two‐dimensional pulsed‐field gel analyses were used together with reciprocal double‐restriction, cross‐hybridization and hybridization fingerprint analysis. These PFG results were confirmed by Smith‐Birnstiel analysis, by Southern hybridization using linking clones and clones of a λ genomic library for the determination of adjacent restriction fragments and by transposon insertion mapping using defined genomic sequences for hybridization. It was thus possible to construct a circular restriction map of the single 9.35 Mbp chromosome of S. aurantiaca based on the endonucleases Asel and Spel. Genetic loci as well as the replication origin were located on the physical map by Southern hybridization using heterologous (derived from Myxococcus xanthus, Escherichia coli and Streptomyces lividans) and homologous probes that are mainly involved in development and ceil motility.
Isolation and characterization of full‐length chromosomes from non‐culturable plant‐pathogenic Mycoplasma‐like organisms
Authors: Neimark Harold , Kirkpatrick Bruce C. 
Published: October 2006

SummaryWe describe the isolation and characterization of full‐length chromosomes from non‐culturable plant‐pathogenic, mycoplasma‐like organisms (MLOs). MLO chromosomes are circular and their sizes (640 to 1185kbp) are heterogeneous. Divergence in the range of chromosome sizes is apparent between MLOs in the two major MLO disease groups, and chromosome size polymorphism occurs among some related agents. MLO chromosome sizes overlap those of culturable mycoplasmas; consequently, small genome size alone cannot explain MLO non‐culturability. Hybridization with cloned MLO‐specific chromosomal and 16S rRNA probes detected two separate chromosomes in some MLO ‘type’ strains. Large DNA molecules that appear to be MLO megaplasmids were also demonstrated. The ability to characterize full‐length chromosomes from virtually any non‐culturable prokaryote should greatly facilitate the molecular and genetic analysis of these difficult bacteria.
SecA protein: autoregulated ATPase catalysing preprotein insertion and translocation across the Escherichia coli inner membrane
Authors: Oliver Donald B. 
Published: October 2006

SummaryRecent insight into the biochemical mechanism of protein translocation in Escherichia coli indicates that SecA ATPase is required both for the initial binding of preproteins to the inner membrane as well as subsequent translocation across this structure. SecA appears to promote these events by direct recognition of the preprotein or preprotein‐SecB complex, binding to inner‐membrane anionic phospholipids, insertion into the membrane biiayer and association with the preprotein translocator, SecY/SecE. ATP binding appears to control the affinity of SecA for the various components of the system and ATP hydrolysis promotes cycling between its different biochemical states. As a component likely to catalyse a rate‐determining step in protein secretion, SecA synthesis is co‐ordinated with the activity of the protein export pathway. This form of negative reguiation appears to rely on SecA protein binding to its mRNA and repressing translation if conditions of rapid protein secretion prevail within the cell. A precise biochemical scheme for SecA‐dependent catalysis of protein export and the details of secA regulation appear to be close at hand. The evolutionary conservation of SecA protein among eubacteria as well as the general requirement for translocation ATPases in other protein secretion systems argues for a mechanistic commonality of all prokaryotic protein export pathways.